Method for the use of botanical extracts in the treatment of viral infections

ABSTRACT

The present disclosure relates generally methods of preparation and use of certain botanical extracts for the treatment of viral infections.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a divisional application that claims priority from U.S.non-provisional application Ser. No. 14/443,767 filed on May 19, 2015,which is the U.S. national stage of PCT Application No.PCT/US2013/071332 filed on Nov. 21, 2013, which claims priority fromU.S. Provisional Application No. 61/778,074 filed Mar. 12, 2013 and U.S.Provisional Application No. 61/729,204 filed Nov. 21, 2012. Allapplications noted hereinabove are hereby incorporated by reference intheir entirety.

FIELD OF TECHNOLOGY

The present disclosure generally relates to antiviral compositions, andmore specifically to the preparation of botanical extracts and their usein the treatment of viral infections, cancer, pain associatedconditions.

BACKGROUND

Poxviruses: Poxviruses are the largest known animal viruses withapproximately 200 distinct genes (Moss, in: Fields Virology, ed. byKnipe and Howley, Philadelphia: Lippincott Williams & Wilkins, 2001, pp.2849-2883). They are DNA viruses that replicate entirely in thecytoplasm. These viruses infect most vertebrates and invertebratescausing a variety of diseases of veterinary and medical importance. ThePoxviridae family has two main subfamilies, the chordopoxyirinae, whichinfect vertebrates, and the entomopoxyirinae, which infect insects. Thechordopoxviruses include the genera Orthopoxvirus, Parapoxvirus,Avipoxvirus, Capripoxvirus, Leporipoxvirus, Suipoxvirus,Mollusciposvirus, and Yatapoxvirus. Each of the chordopoxviruses has arestricted and specific host array. Humans are the sole hosts of twopoxviruses, variola virus (smallpox virus) and molluscum contagiosumvirus: however many members of Orthopoxvirus, Parapoxvirus, andYatapoxvirus are zoonotic, i.e., can infect both animals and humans.Monkeypox virus, an Orthopoxvirus, is a significant zoonotic threat tohumans. Vaccinia virus is the virus used in the variola virus vaccine,and it is widely used as a model poxvirus in the laboratory. Variolavirus, monkeypox virus and vaccinia virus are members of theOrthopoxvirus genus.

In humans, smallpox is a serious, highly contagious, and frequentlyfatal infectious disease for which there is no specific treatment, andfor which the only prevention is vaccination. Two clinical forms ofsmallpox have been described variola minor and variola major, with thevariola major form of smallpox being the more common and severe. Thereare four types of variola major smallpox: ordinary (the most frequent);modified (mild and occurring in previously vaccinated persons); flat;and hemorrhagic. Overall, variola major has a case-fatality rate ofabout 30%.

The most virulent form of smallpox, hemorrhagic smallpox, destroys thelinings of the throat, stomach, intestines, rectum, and vagina andcauses black, unclotted blood to ooze from the mouth and other bodyorifices. Because hemorrhagic smallpox has a much shorter incubationperiod than other forms of smallpox, it is likely not to be initiallyrecognized as smallpox when first presented to medical care. As such,most victims die prior to a correct diagnosis, often before they arequarantined. Smallpox vaccination also does not provide much protection,if any, against hemorrhagic smallpox since hemorrhagic smallpox causesdeath of 94% of vaccinated patients. Hemorrhagic smallpox causes deathin 99% of unvaccinated patients.

Herpes simplex virus 1 (HSV-1) infection occurs in nearly 58 percent ofthe United States population and from 60-95 percent of adults worldwide.Approximately 100 million episodes of recurrent cold sores occur yearlyin the US alone, and studies have shown that HSV-1 infection increasesin prevalence with age. HSV-1 infection is transmitted primarily throughmucocutaneous sites via saliva, respiratory droplets, or secretions.HSV-1 infection occurs predominantly at oral sites and has shown anincreased prevalence in infection at genital sites. It can cause rash,papules, or vesicles following primary infection, and is known to residepermanently in the nervous system of the infected host. During latency,the viral genomes persist as circular, extrachromosomal episomes and theviral lytic genes are repressed. Periodically, the viral genomes in someof the neurons reactivate and the virus travels down the axon to theoriginal site of infection, where it can cause recurrent diseaseresulting in skin ulcerations, neuralgia, and pain. It is the ability tocause an acute infection as well as to repeatedly reactivate and causedisease that is responsible for significant morbidity in humans. Inaddition to causing cold sores (herpes labialis) and more severe oraldisease (herpes gingivitis), HSV-1 also causes herpes stromal keratitis,which is the leading cause of infectious blindness in the US,encephalitis in normal patients, and disseminated infections in immunecompromised individuals.

Although anti-herpes virus therapies are available, their effectivenessis limited and variable. Clearly development of novel and more effectivetherapies for treating herpes associated infections would have a majorhealth impact in the US and worldwide. In addition, it is leasable thatnew therapeutic approaches developed for HSV-1 could translate totreatment of other herpes family members including HSV-2, which cancause severe morbidity and neonatal mortality. There is no availablevaccine for protection against herpesvirus infection, and currenttreatments include nucleoside analogs which target DNA replication ordocosonal which is thought to affect viral membrane fusion. Antiviraldrugs can reduce the severity and duration of lesions, but these do notprevent reactivation and do not lead to the elimination of latent viralgenomes. Typical infections associated with HSV1 have an averageduration from prodrome or erythema stage to healing of 6.1 to 7.9 days.For nucleoside analogs such as acyclovir or valacyclovir, clinicalstudies suggest that these treatments reduce the duration of lesionhealing by 0.5-1.1 days. For docosonal, studies suggest an average 18hour reduction in healing time. Moreover, drag-resistance is common andrenders the current antivirals ineffective.

Another herpes virus member is Varicella Zoster Vims (VZV), thecausative agent of shingles. Due to a worldwide increase in elderly andimmunocompromised individuals, VZV is seen as a “re-emerging” infectionof the twenty-first century. Development of a novel therapeutic is ofmedical importance because VZV is a major cause of morbidity,specifically postherpetic neuralgia, in the aging and immunocompromisedpopulation worldwide. Acyclovir and derivatives (fameyclovir,valacyclovir) which inhibit viral genome replication, are commonly usedprescription drugs in the treatment of herpes virus infections buttreatments produce a moderate reduction in postherpetic neuralgia atbest. In addition, the potential for the development of drug resistancecan render these medications ineffective.

Additional herpes virus members pose significant threats to the humanpopulation including Epstein Barr virus, human herpes 8 (the causativeagent of Kaposi's sarcoma) and cytomegalovirus. Epstein Barr virus inparticular is often associated with the development of jaw and neckcancers.

Several hundred species of human papillomavirus (HPV) have beenidentified. Specific species tend to be associated with human diseaseincluding HPV 16, 18, 31, and 45 as causative agents for cervical, anal,vulvar, vaginal, penile and head/neck cancer; and HPV 1 and 2. as commoncausative agents of warts on the hands and feet. Polyomaviruses arerelated to papillomaviruses and include species such as SV40, JC and BKvirus.

Genital human papillomavirus is the most common sexually transmittedinfection. There are more than 40 HPV types that can infect the genitalareas of males and females. These HPV types can also infect the mouthand throat. Most people who become infected with HPV do not even knowthey have it. Certain strains of HPV can cause precancerous changes incells in the area where the infection occurs. The infection can lead todifferent types of cancers, including cervical cancer in women. About 1%of sexually active adults in the U.S. have genital warts at any giventime. Each year, about 12,000 women get cervical cancer in the U.S.Almost all of these cancers are HPV-associated. Other cancers that canbe caused by HPV are less common than cervical cancer, but areincreasing. Each year in the U.S., there are about: 1,500 women who getHPV-associated vulvar cancer, 500 women who get HPV-associated vaginalcancer, 400 men who get HPV-associated penile cancer, 2,700 women and1,500 men who get HPV-associated anal cancer, 1,500 women and 5,600 menwho get HPV-associated oropharyngeal cancers (cancers of the back ofthroat including base of tongue and tonsils).

Inflammatory responses may also result from various viral infections,inflammation is part of a complex biological response of vasculartissues directed against harmful stimuli, such as pathogens, damagedcells, or irritants. The classical signs of acute inflammation are pain,heat, redness, swelling, and loss of function Inflammation is aprotective attempt by the body to remove the injurious stimuli and toinitiate the healing process, inflammation can be classified as eitheracute or chronic. Acute inflammation is the initial response of the bodyto harmful stimuli and is achieved by the increased movement of plasmaand leukocytes from the blood into the injured tissues. A cascade ofbiochemical events propagates and matures the inflammatory response,involving the local vascular system, the immune system, and variouscells within the injured tissue. Prolonged inflammation, known aschronic inflammation, leads to a progressive shift in the type of cellspresent at the site of inflammation and is characterized by simultaneousdestruction and healing of the tissue from the inflammatory process.Cytokines are several different types of substances that are produced bycells within the immune system that relay signals between the immunesystem cells. Pro-inflammatory cytokines are created primarily by immunecells that are engaged in the process of amplifying inflammatoryreactions as a means of dealing with a health threat to the body. Byrelaying messages between the cells, these cytokines help to trigger theimmune system's rate of response to the threat. There is abundantevidence that certain pro-inflammatory cytokines such as IL-1 β, IL-6,and TNF-α are involved in the process of pathological pain.

Based on the prevalence of these and other diseases and infections,there is a continuing need for new anti-viral, anti-cancer,anti-pruritic (anti-itch) analgesic (anti-pain), and anti-inflammatorytreatments.

SUMMARY

The present disclosure relates generally to the preparation and use ofcertain botanical extracts and therapeutic compositions comprising thesame, in the treatment of viral infections such as poxvirus infections,including, but not limited to, variola virus, vaccinia virus, Monkeypoxvirus, Cowpox virus, Ectromelia virus, Orf virus, Canarypox virus,Myxoma virus, and Moiluscum contagiosum; herpesvirus infections,including, but not limited to herpes simplex virus-1 (HSV1), herpessimplex virus-2 (HSV2), Human herpes 8 and Equine herpes virus (EHV);Varicella-Zoster virus (VZV), Cytomegalovirus, Epstein-Barr virus,Kaposi's Sarcoma Associated herpesvirus (HHV8), Human papillomavirus(including various serotypes of HPV) and polyomavirus infections(including JC and BK viruses); cancerous and pre-cancerous lesions(including, but not limited to cervical cancer, squamous cell carcinomaand actinic keratosis); cancer associated with a papilloma virus orEpstein Barr virus infection, including actinic keratosis, squamous cellcarcinoma, cervical cancer jaw, neck, throat, penile and anal cancer;and, pain and itch associated with diseases in general.

Botanical extracts, in accordance with the present disclosure, compriseextracts of one or more plant tissues. In various embodiments, botanicalextracts include at least one extract of a carnivorous plant species, invarious embodiments of the present disclosure, botanical extractsinclude at least one extract of the Sarracenia, Nepenthes, Melissa,Lavandula, Glycyrrhiza, Eleutherococcus, Hypericum, Darlingtonia,Heliamphora, Roridula, Drosera, Dionaea, Aldrovanda, Drosophyllum,Triphyophyllum, Catopsis, Brocchinia, Paepalanthus, Utricularia,Genlisea, Pinguicula, Ibicella, Byblis, Philcoxia, Stylidium and/orCephalotus genera. Such botanical extracts will be referred to herein as“Genus Extracts.”

In various embodiments of the present disclosure, botanical extractscomprise extracts from plant species including, but not limited to, S.purpurea, S. alata, S. flava, S. leucophylla, S. rubra, S. alabamensis,S. jonesii, S. minor, S. oreophila, S. psittacina, N. Judith Finn, N.speciabilis x N. ventricosa (hybrid), N. veitchii, N. eymae, N. fusca, Kchaniana, N. macrophylla, M. officinalis, L. officinalis, G. glabra, E,senticosus, H, perforatum, D. californicum, R. gorgonias, D. intermedia,D. cuneifolia, D. muscipula, A. vesiculosa, N. rafflesiana, D.lusitanicum, T. peltatum, C. berteroniana, B. hechtiodes, B. tatei, P.celsus, P. acantholimon, U. gibba, U. geminiscapa, G. repens, P.potosiensis, I. lutea, B. liniflora and C. follicularis. Such botanicalextracts will be referred to herein as “Species Extracts.”

In various aspects, the present disclosure provides methods of makingbotanical extracts. The extracts of the present disclosure may becombined and/or formulated with any food-grade or pharmaceuticallyacceptable excipient, such as to provide various therapeuticcompositions or pharmaceutical formulations, as further describedherein. As used herein, a therapeutic composition includes anycomposition or formulation intended for pharmaceutical,over-the-counter, direct-to-consumer, and all other appropriate retailand wholesale markets for human or animal consumption.

The present disclosure further provides methods of inhibiting viralreplication using various botanical extracts. In various embodiments,the present disclosure provides methods of treating virus infectionssuch as a poxvirus infection, (e.g., a vaccinia virus or a variola virusinfection); and methods of reducing viral load, or reducing the time toviral clearance, or reducing morbidity or mortality in the clinicaloutcomes, in patients suffering from a pox viral infection. The presentdisclosure further provides methods of reducing the risk that anindividual will develop a pathological poxvirus infection, such as avaccinia virus infection or a variola virus infection, which hasclinical sequelae. The methods generally involve administering atherapeutically effective amount of at least one botanical extractand/or botanical extract composition, either alone or in combinationwith other therapeutic compositions, for the treatment of a viralinfection, particularly a pox viral infection.

As used herein, the term “poxvirus” includes any member of the familyPoxyiridae, including, but not limited to, any member of theOrthopoxvirus genus, including variola (smallpox) virus, vaccinia virus,camelpox, cowpox, ectromelia, monkeypox, racoonpox, skunkpox, taterapox,Uasin Gishu, and volepox; any member of the Parapoxvirus genus; anymember of the Avipoxvirus genus; any member of the Capripoxyinus genus;any member of the Leporipoxvirus genus; any member of the Suipoxvirusgenus; any member of the Molluscipoxvirus genus; any member of theYatapoxvirus genus; and any member of the Entomopoxvirus A, B, or Cgenus.

The term “poxvirus” further includes naturally-occurring (e.g.,wild-type) poxvirus; naturally-occurring poxvirus variants; and poxvirusvariants generated in the laboratory, including variants generated byselection, variants generated by chemical modification, and geneticallymodified variants (e.g., poxvirus modified in a laboratory byrecombinant DNA methods).

In various aspects, the present disclosure comprises the use of abotanical extract composition comprising at least one botanical extractof Sarracenia, Nepenthes, Melissa, Lavandula, Glycyrrhiza,Eleutherococcus, Hypericum, Darlingtonia, Heliamphora, Roridula,Drosera, Dionaea, Aldrovanda, Drosophyllum, Triphyophyllum, Catopsis,Brocchinia, Paepalanthus, Utricularia, Genlisea, Pinguicula, Ibicella,Byblis, Philcoxia, Stylidium or Cephalotus genera in the treatment ofviruses including, but not limited to, variola virus, vaccinia virus,Monkeypox virus, Cowpox virus, Ectromelia virus, Orf virus, Canarypoxvirus, Myxoma virus, and Molluscum contagiosum.; herpesvirus infections,including, but not limited to herpes simplex virus-1 (HSV1), herpessimplex virus-2 (HSV2), Human herpes 8 and Equine herpes virus (EHV);Varicella-Zoster virus (VZV), Cytomegalovirus, Epstein-Barr virus,Kaposi's Sarcoma Associated herpesvirus (HHV8), Human papillomavirus(including various serotypes of HPV), polyomavirus (including JC and BKviruses), and in the treatment of any associated herpes virus inducedcancers.

In various embodiments, certain botanical extracts including extractsfrom one or more of the genera Nepenthes, Drosera, Dionaea, Darlingtoniaand Pinguicula, demonstrate antiviral activity. Such botanical extractswill be referred to herein as “Genus II Extracts.” In variousembodiments, S. purpurea exhibited antipoxvirus activity. However,various combinations of two or more botanical extracts showed antiviralactivity that was: (1) less than the predicted additive activity (i.e.,a “negative” effect); (2) equal to the predicted additive activity(i.e., an “additive” effect); or, (3) unexpectedly greater than thepredicted additive activity (i.e., a “synergistic” effect),demonstrating unpredicted synergistic combinations of botanical extractsnot heretofore known or expected in the art.

In various embodiments, the present disclosure comprises a therapeuticcomposition comprising a botanical extract and at least onepharmaceutically acceptable excipient.

In certain embodiments, the present disclosure provides a method fortreating or preventing a viral infection in an animal or humancomprising administering a botanical extract of one or more of thegenera Sarracenia and Nepenthes botanical extracts (referred to hereinas “Genus III Extracts”), formulated in a pharmaceutically acceptabletransdermal driving carrier/gel, to an animal or human.

In various embodiments, botanical extracts provided herein, may haveutility in the treatment, prevention, and/or other effect in relation topoxvirus infections, including, but not limited to, variola virus,vaccinia virus, Monkeypox virus. Cowpox virus, Ectromelia virus, Orfvirus, Canarypox virus, Myxoma virus, and Molluscum contagiosum.;herpesvirus infections, including, but not limited to herpes simplexvirus-1 (HSV1), herpes simplex virus-2 (HSV2), Human herpes 8 and Equineherpes virus (EHV); Varicella-Zoster virus (VZV), Cytomegalovirus,Epstein-Barr virus, Kaposi's Sarcoma Associated herpesvirus (HHV8),Human papillomavirus (including various serotypes of HPV) andpolyomavirus infections (including JC and BK viruses) (collectively, the“Target Viruses”). Accordingly, the present disclosure provides methodsof treating virus infections, such as a poxvirus infection, and methodsof reducing viral load, or reducing the time to viral clearance, orreducing morbidity or mortality in the clinical outcomes, in patientssuffering from a viral infection. In various embodiments, the presentdisclosure provides methods for treating and/or preventing an infectionof the Target Viruses in an animal or human comprising administering abotanical composition described herein as a liquid, or formulated in apharmaceutically acceptable transdermal driving carrier/gel, to theanimal or human.

In various embodiments, the present disclosure provides a method forprophylactic or therapeutic treatment of cancer in an animal or humancomprising administering one or more botanical extracts in apharmaceutically acceptable transdermal driving carrier/gel to an animalor human.

In various embodiments, a method for prophylactic or therapeutictreatment of cancer in an animal or human comprises administering atherapeutically effective amount of one or more Genus III Extracts.

In certain embodiments, the present disclosure provides a method forprophylactic or therapeutic treatment of cervical dysplasia in a patientin need thereof, comprising administering a therapeutically effectiveamount of one or more botanical extracts, either alone or with apharmaceutically acceptable transdermal driving carrier/gel. In variousembodiments, a method for prophylactic or therapeutic treatment ofcervical dysplasia in a patient in need thereof comprises administeringa therapeutically effective amount of one or more Genus III Extracts,either alone or with a pharmaceutically acceptable transdermal drivingcarrier/gel.

In various embodiments, the present disclosure provides a method oftherapeutic treatment of itching (anti-pruritic) in a patient in needthereof, comprising administering a therapeutically effective amount ofone or more Genus III Extracts, either alone or with a pharmaceuticallyacceptable transdermal driving carrier/gel. In certain embodiments, amethod of therapeutic treatment of itching (anti-pruritic) in a patientin need thereof comprises administering a therapeutically effectiveamount of one or more Genus III Extracts, either alone or with apharmaceutically acceptable transdermal driving carrier/gel.

In various embodiments, the present disclosure provides a method oftherapeutic treatment of pain (analgesic) in a patient in need thereof,comprising administering a therapeutically effective amount of one ormore Genus III Extracts, either alone or with a pharmaceuticallyacceptable transdermal driving carrier/gel. In various embodiments, amethod of therapeutic treatment of pain (analgesic) in a patient in needthereof comprises administering a therapeutically effective amount ofone or more Genus III Extracts, either alone or with a pharmaceuticallyacceptable transdermal driving carrier/gel.

In various embodiments, the present disclosure provides a method for theprophylactic or therapeutic treatment of precancerous lesions or cancer,including actinic keratosis, squamous cell carcinoma, cervicaldysplasia, cervical cancer, jaw/neck/throat cancer, anal cancer andpenile cancer, comprising the administration of one or more botanicalextracts, either alone or with a pharmaceutically acceptable transdermaldriving carrier/gel. In various embodiments, a composition comprising aGenus III Extract and/or a botanical formulation as described herein,alone or with a pharmaceutic ally acceptable transdermal drivingcarrier/gel, is used for the prophylactic or therapeutic treatment ofprecancerous lesions or cancer including actinic keratosis, squamouscell carcinoma, cervical dysplasia, cervical cancer, jaw/neck/throatcancer, anal cancer and penile cancer.

In various embodiments, the present disclosure includes methodscomprising administering one or more botanical extracts and/orpharmaceutical formulation thereof, to a patient in need of treatment,for example, a patient infected with a virus, or to inhibit virusreplication or infection of a cell in a patient, or to treat a diseaseor condition associated with such virus replication or infection of acell. Although not wishing to be bound to any theories of action,botanical extracts of the present disclosure are believed to block viralreplication. For example, Sarracenia spp. is believed to block viral RNAsynthesis. Melissa spp. is believed to block viral attachment to thecell.

In various embodiments, a botanical extract composition comprises one ormore botanical extracts. For example, in certain embodiments, abotanical extract composition comprises at least one of Sarraceniapurpurea, Melissa officinalis, Lavandula officinalis, Glycyrrhizaglabra, Eleutherococcus senticosus and/or Hypericum perforatum. Suchbotanical extracts will be referred to herein as “Species II Extracts.”

In various embodiments, botanical extract compositions comprising one ormore of Species II Extracts provide additive or synergistic antiviralactivity.

In various aspects, a method for preparing a liquid extract from a plantmaterial chosen from Sarracenia, Nepenthes, Melissa, Lavandula,Glycyrrhiza, Eleutherococcus, Hypericum, Darlingtonia, Heliamphora,Roridula, Drosera, Dionaea, Aldrovanda, Drosophyllum, Triphyophyllum,Catopsis, Brocchinia, Paepalanthus, Utricularia, Genlisea, Pinguicula,Ibicella, Byblis, Philcoxia, Stylidium and Cephalotus, said methodcomprising, obtaining fresh plant material; washing and air drying saidplant material: combining said plant material with a liquid comprisingat least one of water, ethanol and glycerol; allowing said liquid toextract said plant material at from about room temperature to simmeringtemperatures to form said liquid extract; and separating said liquidextract from said plant material, is provided.

In various aspects, a method for extracting the roots or leaves from thebotanical Sarracenia purpurea comprising, obtaining fresh plantmaterial; washing and air drying said plant material; combining saidplant material with a liquid comprising at least one of ethanol, waterand glycerol, wherein the combination of plant material to liquid isfrom about 1:2. up to about 1:5 grams of plant material to millilitersof liquid, and wherein said ethanol is from about 45% to about 70% ofsaid liquid and said glycerol is from about 0% to about 10% in saidliquid; allowing said liquid to extract said plant material at roomtemperature for 2-60 days, or for 1-3 hours in simmering conditions whensaid liquid is only water; separating said liquid from said plantmaterial; and optionally filtering said liquid to remove largeparticulates, is provided. In various embodiments, said liquid comprises63% 190 proof ethanol, 32% water and 5% glycerol. In variousembodiments, said liquid comprises from about 50% to about 80% ofglycerol, with the remainder being water and no ethanol. In someembodiments, said liquid consists of only water. In various embodiments,said liquid comprises 25% water and 75% glycerol.

In various aspects, a method of extracting the aerial portions of thebotanical Melissa officinalis comprising, removing leaves from stems;washing and air drying said leaves; blending said leaves at a ratio of1:2-1:5 grams of plant material to milliliters of liquid, said liquidcomprising distilled water and glycerol, said glycerol present at fromabout 50% to about 80% in said liquid; allowing said liquid to extractsaid leaves for 2 to 40 days at room temperature; separating said liquidfrom said plant material; and optionally filtering said liquid to removelarge particulates, is provided.

In various aspects, a method for extracting the flowering stems of thebotanical Lavandula officinalis comprising, obtaining fresh Lavandulaofficinalis plants; removing flowers from stems; blending said flowersat a ratio of 1:2-1:5 grams of plant material to milliliters of liquid,said liquid comprising distilled water and glycerol, said glycerolpresent at from about 50% to about 80% in said liquid; allowing saidliquid to extract said leaves for 2 to 40 days at room temperature;separating said liquid from said plant material; and optionallyfiltering said liquid to remove large particulates, is provided.

In various aspects, a method for extracting the flowering stems of thebotanical Hypericum perforatum comprising, obtaining fresh Hypericumperforatum plants; removing flowers and leaves from stems; blending saidflowers and leaves at a ratio of 1:2-1:5 grams of plant material tomilliliters of liquid, said liquid comprising 190-proof ethanol,distilled water and glycerol, said ethanol present at from 40% to 70%and said glycerol present at 0% to 5%; allowing said liquid to extractsaid leaves for 2 to 40 days at room temperature; separating said liquidfrom said plant material; and optionally filtering said liquid to removelarge particulates, is provided.

In various aspects, a method for extracting the botanicalEleutherococcus senticosus comprising, obtaining fresh Eleutherococcussenticosus plants; blending said plants at a ratio of 1:2-1:5 grams ofplant material to milliliters of liquid, said liquid comprising190-proof ethanol, distilled water and glycerol, said ethanol present atfrom 25% to 60% and said glycerol present at 0% to 5%; allowing saidliquid to extract said leaves for 2 to 40 days at room temperature;separating said liquid from said plant material; and optionallyfiltering said liquid to remove large particulates, is provided.

In various aspects, a method for extracting the botanicalEleutherococcus senticosus comprising, obtaining a powdered extract ofEleutherococcus senticosus obtained from a 10:1 ethanol/waterextraction; combining said powdered extract at a ratio of 1:10 grams ofpowdered extract to milliliters of liquid, said liquid comprising 25% to60% ethanol and 0% to 5% glycerol; mixing for 14 days at roomtemperature; and filtering said powder from said liquid, is provided.

In various aspects, a method for extracting the botanical Glycyrrhizaglabra comprising, obtaining fresh Glycyrrhiza glabra plants: blendingsaid plants at a ratio of 1:2-1:5 grams of plant material to millilitersof liquid, said liquid comprising 190-proof ethanol, distilled water andglycerol, said ethanol present at from 10% to 50% and said glycerolpresent at 0% to 5%; allowing said liquid to extract said leaves for 2to 40 days at room temperature; separating said liquid from said plantmaterial; and optionally filtering said liquid to remove largeparticulates, is provided.

In various aspects, a method for extracting the botanical Glycyrrhizaglabra comprising, obtaining a powdered extract of Glycyrrhiza glabraobtained from a 8:1 ethanol/water extraction; combining said powderedextract at a ratio of 1:10 grams of powdered extract to milliliters ofliquid, said liquid comprising 10% to 50% ethanol and 0% to 5% glycerol;mixing for 14 days at room temperature; and filtering said powder fromsaid liquid, is provided.

In various embodiments, a therapeutic composition comprises at least oneliquid extract from any of claims 1-13.

In various embodiments, an aqueous botanical extract mixture comprises:50% extract of Sarracenia purpurea (equating to 50 viral inhibitoryunits (VIU)/ml); 12% Melissa officinalis (equating to 80 viralinhibitory units (VIU)./ml); 20% Lavandula officinalis (equating to 20viral inhibitory units (VIU)/ml); 5% Glycyrrhiza glabra (equating to 40viral inhibitory units (VIU)/ml): 5% Eleutherococcus senticosus(equating to 40 viral inhibitory units (VIU)/ml); and optionally, 8%Hypericum perforatum (equating to 30 viral inhibitory units (VIU)/ml),wherein each of said extracts are prepared by the methods of claims1-13. In various aspects, an aqueous botanical extract mixturecomprises: 40-100% extract of Sarracenia purpurea, 5-30% Melissaofficinalis, 5-35% Lavandula officinalis, 2-30% Glycyrrhiza glabra,2-20% Eleutherococcus senticosus and/or 5-20% Hypericum perforatum.

In various aspects, a synergistic botanical extract mixture comprisesSarracenia purpurea, Melissa officinalis, Lavandula officinalis,Glycyrrhiza glabra, Eleutherococcus senticosus and/or Hypericumperforatum.

In various embodiments, the use of a therapeutic composition for dermalor epithelial topical therapy, wherein said formulation comprises atleast one extract of any one of claims 1-18 for a suspension in atransdermal base, is provided. In some embodiments, said transdermalbase is chosen from the group consisting of VERSABASE, LIPODERM,PENTRAVAN, Pluronic Lecithin Organogel (PLO), and mixtures thereof. Infurther embodiments, the use of a therapeutic composition of claim 19for transdermal drug delivery on the cervix or vaginal tract of apatient in need of therapy, is provided.

In embodiments, a therapeutic composition comprises, at least oneextract in accordance with any one of claims 1-13; and at least onepharmaceutically acceptable excipient. In some embodiments, said atleast one extract and said at least one pharmaceutically acceptableexcipient are in a ratio of 50/50, In further embodiments, said at leastone extract and said at least one pharmaceutically acceptable excipientare in a ratio of 75/25. In some embodiments, said pharmaceuticallyacceptable excipient is chosen from the group consisting of ammoniumacryloyldimethyltaurate/VP Copolymer, aloe vera, allantoin, fatty acids,fatty alcohols, fatty acid esters, monoglycerides, diglycerides,triglycerides, glycerin, lecithin, gums, polymers, sorbitan fatty acidesters, alkoxylated fatty acids, alkoxylated fatty acid esters, andmixtures thereof.

In various aspects, a therapeutic composition comprises an extract of atleast one of Sarracenia, Nepenthes, Melissa, Lavandula, Glycyrrhiza,Eleutherococcus, Hypericum, Darlingtonia, Heliamphora, Roridula,Drosera, Dionaea, Aldrovanda, Drosophyllum, Triphyophyllum, Catopsis,Brocchinia, Paepalanthus, Utricularia, Genlisea, Pinguicula, Ibicella,Byblis and Cephalotus genera plant; and a fatty acid component. Invarious embodiments, the therapeutic composition of claim 26 comprises apitcher plant extract and a fatty acid component,

In various embodiments, a method for inhibiting the replication of apoxvirus comprises exposing said poxvirus to at least one extract of anyone of claims 1-18. In some embodiments, said poxvirus is chosen fromthe group consisting of orthopoxvirus, parpoxvirus, avipoxvirus,capripoxvirus, leporipoxvtrus, suipoxvirus, molluscipoxvirus andyatapoxvirus.

In various embodiments, a method for inhibiting the replication of humanpoxvirus pathogens comprises exposing said poxvirus pathogen to at leastone extract from any one of claims 1-18. In some embodiments, saidpoxvirus pathogen is chosen from the group consisting of vaccinia virus,monkeypox virus, variola virus, and molluscum contagiosum virus.

In various embodiments, a method of inhibiting the replication of animalpoxvirus pathogens comprises exposing said animal poxvirus pathogen toat least one extract of any one of claims 1-18.

In various embodiments, a method of inhibiting the replication of herpesvirus family members comprises exposing said herpes virus to at leastone extract of any one of claims 1-18. In some embodiments, said herpesviruses is selected from the group consisting of herpes simplex 1,herpes simplex 2, varicella-zoster virus, Epstein-Barr virus,cytomegalovirus and human herpes 8. In further embodiments, said herpesvirus is an animal herpes virus. In yet further embodiments, said animalherpes virus is equine herpes virus-1.

In various embodiments, a method of inhibiting the replication ofpapilloma and polyomavirus family members comprises exposing saidpapilloma or polyomavirus family member to at least one extract of anyone of claims 1-18.

In various embodiments, a method of inhibiting the replication of humanpapillomavirus pathogens comprises exposing said human papillomaviruspathogen to at least one extract of any one of claims 1-18. In someembodiments, said human papillomavirus pathogen is any one of HPV 2, 6,7, 11, 14, 16, 18, 31, 32, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59 and63.

In various embodiments, a method of inhibiting the replication of humanpolyomavirus pathogens comprises exposing said polyomavirus pathogen toat least one extract of any one of claims 1-18. In some embodiments,said pathogen is any one of JC and BK viruses.

In various embodiments, a method for inducing cellular senescence andapoptosis in human papillomavirus-induced cancer cells comprisesexposing said cancer cells to at least one extract of any one of claims1-18,

In various embodiments, a method for prophylactic or therapeutictreatment of human poxvirus infections comprises exposing said humanpoxvirus to any therapeutic composition of any one of claims 19, and22-27. In some embodiments, the viral infection is selected from thegroup consisting of vaccinia virus, monkeypox virus, variola virus andmolluscum contagiosum virus.

In various embodiments, a method for prophylactic or therapeutictreatment of animal poxvirus infections comprises exposing said animalpoxvirus to any extract of claims 1-18.

In various embodiments, a method for prophylactic or therapeutictreatment of animal poxvirus infections comprises exposing said animalpoxvirus to any therapeutic composition of claims 19, and 22-27.

In various embodiments, a method for prophylactic or therapeutictreatment of human herpes virus infections comprises exposing said humanherpes virus to any extract of claims 1-18 or any therapeuticcomposition of claims 19, and 22-27. In some embodiments, the viralinfection is selected from the group consisting of herpes simplexvirus-1 (HSV1), herpes simplex virus-2 (HSV2), herpes varicella-zoster,Epstein-Barr virus, cytomegalovirus and human herpes 8.

In various embodiments, a method for prophylactic or therapeutictreatment of animal herpes virus infections comprises exposing saidanimal herpes virus to any extract of claims 1-18 or any therapeuticcomposition of claims 19, and 22-2.7. In some embodiments, the viralinfection is equine herpes virus-1.

In various embodiments, a method for prophylactic or therapeutictreatment of human papillomavirus infections comprises exposing saidhuman papillomavirus to any extract of claims 1-18 or any therapeuticcomposition of claims 19, and 22-27, In some embodiments, the viralinfection is at least one of human papillomaviruses HPV 2, 6, 7, 11, 14,16, 18, 31, 32, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59 and 63.

In various embodiments, a method for prophylactic or therapeutictreatment of human polyomavirus infections comprises exposing said humanpolyomavirus to any extract of claims 1-18 or any therapeuticcomposition of claims 19, and 22-27. In some embodiments, the viralinfection is one of human J C and BK viruses.

In various embodiments, a method for prophylactic or therapeutictreatment of pre-cancer or cancer, wherein the pre-cancer or cancer isactinic keratosis or squamous cell carcinoma, comprises exposing saidpre-cancer or cancer to any extract of claims 1-18 or any therapeuticcomposition of claims 19, and 22-27.

In various embodiments, a method for prophylactic or therapeutictreatment of pre-cancer or cancer, wherein the pre-cancer or cancer iscervical dysplasia or cervical cancer, comprises exposing saidpre-cancer or cancer to any extract of claims 1-18 or any therapeuticcomposition of claims 19, and 22-27.

In various embodiments, a method for prophylactic or therapeutictreatment of cancer, wherein the cancer is anal cancer, comprisesexposing said cancer to any extract of claims 1-18 or any therapeuticcomposition of claims 19, and 22-27.

In various embodiments, a method for prophylactic or therapeutictreatment of cancer, wherein the cancer is oropharyngeal cancer,laryngeal papillomas, or oral papillomas, comprises exposing said cancerto any extract of claims 1-18 or any therapeutic composition of claims19, and 22-27.

In various embodiments, a method for prophylactic or therapeutictreatment of warts, wherein the warts include plantar warts, commonwarts and anogenital warts, comprises exposing said warts to any extractof claims 1-18 or any therapeutic composition of claims 19, and 22-27.

In various embodiments, a method for prophylactic or therapeutictreatment of cancer, wherein the cancer is Kaposi's Sarcoma, comprisesexposing said cancer to any extract of claims 1-18 or any therapeuticcomposition of claims 19, and 22-27.

In various embodiments, a method for prophylactic or therapeutictreatment of pre-cancer or cancer associated with herpes virusinfections, comprises exposing said pre-cancer any extract of claims1-18 or any therapeutic composition of claims 19, and 22-27. In someembodiments, the viral-induced cancer is one of Epstein-Barr virus andHuman herpes 8 virus.

In various embodiments, a method for prophylactic or therapeutictreatment of pre-cancer or cancer associated with papillomavirusinfections comprises exposing said pre-cancer or cancer to any extractof claims 1-18 or any therapeutic composition of claims 19, and 22-27.In some embodiments, the viral-induced cancer is associated with any oneof human papilloma viruses HPV 16, 18, 31 and 45.

In various embodiments, a method for therapeutic treatment of itching(anti-pruritic) in a patient in need thereof, comprises administering tosaid patient a therapeutically effective amount of a botanical extractof any one of claims 1-18.

In various embodiments, a method for therapeutic treatment of pain(analgesic) in a patient in need thereof, comprises administering tosaid patient a therapeutically effective amount of a botanical extractof any one of claims 1-18.

In various embodiments, said plant is selected from the group consistingof S. elata, S. flava, S. leucophylla and S. rubra. In some embodiments,said plant is selected from the group consisting of N. veitchii, N.spectrabilis x ventricosa, N. eymae, N. Judith Finn, N. chaniana, N.fusca and N. macrophylla. In further embodiments, said plant is selectedfrom the group consisting of Drosera spp., Dionaea spp., Darlingtoniaspp., and Pinguicula spp.

In various embodiments, a topical therapeutic composition comprises anextract of claims 1-18 or a therapeutic composition of claims 19, and22-21.

In various embodiments, an oral formulation comprises an extract ofclaims 1-18 or a therapeutic composition of claims 19, and 22-27.

In various embodiments, a suppository comprises an extract of claims1-18 or a therapeutic composition of claims 19, and 22-27.

In various embodiments, a method for administering an extract of claims1-18 or a therapeutic composition of claims 19, and 22-27 consisting ofadministering said extract or therapeutic composition through at leastone of oral, buccal, rectal, intranasal, parenteral, intraperitoneal,intradermal, subcutaneous, intramuscular, transdermal, inhalation andintratracheal routes, is provided.

In various aspects, a method for the standardization of botanicalextracts for antiviral activity and therapeutic value comprising, adetermination and measurement of the antiviral units per milliliter, isprovided.

In various aspects, an aqueous mixture comprising, 50% extract ofSarracenia purpurea (equating to 50 viral inhibitory units (VIU)/ml);12% Melissa officinalis (equating to 80 viral inhibitory units(VIU)/ml); 20% Lavandula officinalis (equating to 20 viral inhibitoryunits (VIU)/ml); 5% Glycyrrhiza glabra (equating to 40 viral inhibitoryunits (VTU)/ml); 5% Eleutherococcus senticosus (equating to 40 viralinhibitory units (VIU)/ml); and optionally, 8% Hypericum perforatum(equating to 30 viral inhibitory units (VIU)/ml), is provided.

In various aspects, a composition comprising at least one of a GenusExtract, is provided.

In various aspects, a composition comprising at least one of a Genus IIExtract, is provided.

In various aspects, a composition comprising at least one of a SpeciesExtracts, is provided.

In various aspects, a composition comprising at least one of a SpeciesII Extract, is provided.

In various embodiments, the composition of claim 78, further comprises apharmaceutically acceptable excipient. In some embodiments, thecomposition of claim 78, further comprises a gel carrier. In furtherembodiments, the composition of claim 78 is aqueous.

In various aspects, a composition comprising at least one of thefollowing botanical extract pairs, Melissa and Sarracena; Melissa andGlycyrrhiza: Melissa and Eleutherococcus; Sarracenia andEleutherococcus; Sarracenia and Glycyrrhiza; Sarracenia and Hypericum;Sarracenia and Lavandula; Eleutherococcus and Glycyrriza; Glycyrhizzaand Lavandula; Eleutherococcus and Hypericum; and Eleutherococcus andLavandula, is provided. In various embodiments, the composition of claim83 further comprises a pharmaceutically acceptable excipient In someembodiments, the composition of claim 83, further comprises a gelcarrier. In further embodiments, the composition of claim 83 is aqueous.

In various aspects, a composition comprising at least one of a Genus IIIExtract, is provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1 C illustrate the effect of a S. purpurea extract on VACVreplication;

FIGS. 2A-2D illustrate the effect of a S. purpurea extract on VACVinduced CPE and protein synthesis;

FIGS. 3A and 3B illustrate the effect of a S. purpurea extract on VACVtranscription in vivo and in vitro;

FIG. 4 illustrates a possible mechanism of action of poxvirustherapeutics;

FIGS. 5A and 5B illustrate a specificity of a S. purpurea extract onOrthopoxvirus;

FIGS. 6A-6E illustrates an effect of a S. purpurea extract on HSV1induced CPE;

FIG. 7 illustrates an effect of a S. purpurea extract on HSV1 proteinaccumulation;

FIG. 8 illustrates an effect of a S. purpurea extract on HSV1replication;

FIGS. 9A-9H illustrate an effect of a S. purpurea extract on VZVreplication;

FIGS. 10A-10D illustrate an effect of a S. purpurea extract on VZVplaque formation;

FIG. 11 illustrates an effect of a S. purpurea extract on VZV proteinaccumulation;

FIG. 12 illustrates an effect of a S. purpurea extract on thereplication of herpes virus family members;

FIGS. 13A-13D illustrate an effect of a S. purpurea extract as atherapeutic for HSV1 infections;

FIGS. 14A-14E illustrate an effect of a S. purpurea extract on polyomavirus SV40 induced CPE (cytopathic effect);

FIG. 15 illustrates an effect of a S. purpurea extract on polyoma virusSV40 protein accumulation;

FIGS. 16A-16E illustrate an effect of a S. purpurea extract on cervicalcancer cells;

FIGS. 17A-17D illustrates an effect of a S. purpurea extract on cervicalcancer cells at higher magnification;

FIGS. 18A and 18B illustrate an effect of a S. purpurea extract on HPVprotein expression in cervical cancer cells;

FIGS. 19A and 19B illustrate an effect of a S. purpurea extract on HPVprotein expression in cervical cancer cells;

FIGS. 20A and 20B illustrate an effect of a S. purpurea extract oncellular p53 protein expression in cervical cancer cells;

FIG. 21 illustrates an effect of a S. purpurea extract on cervicalcancer cell replication;

FIG. 22 illustrates a process for standardization of a botanicalextract;

FIGS. 23A-23C illustrate structures of betulin and betulin derivatives;

FIGS. 24A-24C illustrate antiviral activity associated with betulin andbetulin derivatives;

FIGS. 25A-25C illustrate antiviral activity screening results forbotanical extracts;

FIGS. 26A and 26B illustrate antiviral activity screening results forbotanical extracts;

FIG. 27 illustrates a specificity of a S. purpurea extract antiviralactivity;

FIG. 28 illustrates a specificity of a M. officinalis extract antiviralactivity;

FIGS. 29A-29D illustrate a specificity of a G. glabra, L. officinalis,H. performatum and E. senticosus extract antiviral activity;

FIGS. 30A-30F illustrate anti-herpes virus activity associated withbotanical extracts;

FIGS. 31A and 31B illustrate synergistic activity associated with abotanical extract;

FIGS. 32A-32E illustrate antiviral activity of Sarracenia species;

FIG. 33 illustrates antiviral activity of Nepenthes species;

FIG. 34 illustrates inhibition of herpes virus CPE by M. officinalis;

FIGS. 35A and 35B illustrate inhibition of HSV1 spread by M.officinalis;

FIG. 36 illustrates inhibition of HSV 1 protein accumulation by M.officinalis;

FIGS. 37A-37C illustrate activity of M. officinalis toward HSV1;

FIG. 38 illustrates M. officinalis extract effect on HSV1 attachment toheparin;

FIGS. 39A and 39B illustrate M. officinalis extract effect on viralfamilies:

FIG. 40 illustrates effects of M. officinalis and G. glabra extracts onpolyoma SV40 virus replication;

FIGS. 41A-41C illustrate effects of therapeutic treatment of VZVinfected subject with a botanical extract formulation;

FIG. 42A-42R illustrate in vitro anti-inflammatory activity associatedwith S. purpurea extracts;

FIGS. 43A-43I illustrate cytokine response of S. purpurea treated PBMCs;

FIGS. 44A-44I illustrate cytokine response of S. purpurea treated PBMCs;and FIGS. 45A-45F illustrate antiviral activity of ethanol and glycerinbotanical extracts.

DETAILED DESCRIPTION

In accordance with the present disclosure, methods making botanicalextracts are disclosed, in addition to various methods of blendingcertain botanical extracts, compounding of various botanical extractsinto therapeutic compositions, and various methods of use of bothbotanical extracts and therapeutic compositions for the treatment andprophylaxis of infections, diseases and symptoms thereof. In variousembodiments, tissue from a plant of a genus and/or species identified inthe present disclosure is used in the preparation of a botanicalextract. In certain embodiments of the present disclosure, one or moreof Species II Extracts, amongst others, can be used in the preparationof a botanical extract.

Botanical extracts in accordance with the present disclosure, functiontherapeutically in an antiviral capacity either individually or inspecific combinations. Select combinations of botanical extracts provideunexpected synergistic antiviral activity. In various embodiments, theviral infection is associated with poxvirus infections, including, butnot limited to, the Target Viruses. As such, the present disclosureprovides methods of treating infections of the Target Viruses andmethods of reducing viral load, or reducing the time to viral clearance,or reducing morbidity or mortality in the clinical outcomes, in patientssuffering from a viral infection.

The present disclosure contemplates the use of one or more of GenusExtracts, Genus II Extracts, Genus III Extracts, Species Extracts andSpecies II Extracts in various embodiments.

In various aspects, botanical extract preparations may undergo qualitycontrol and/or standardization wherein the antiviral activity of eachextract is measured and/or adjusted prior to and after preparation ofthe aqueous blend. As used herein, 1 VIU is defined as a viralinhibitory unit and is the quantity of an extract, composition, orformulation required to inhibit viral replication by 50%. In variousaspects, the antiviral activity of a botanical extract, botanicalextract composition, or therapeutic composition is measured using aplaque reduction technique in a cell culture system and titration of theextract, composition, or formulation, as described elsewhere herein. Invarious embodiments, the antiviral activity of an extract, composition,or formulation may be expressed in any suitable manner, such as VIU/ml.

As used herein, the term “percent plaque formation” and variationsthereof refers to the proportion of expected plaque formation in an invitro biological activity assay to assess the antiviral effects of acompound, extract, composition, formulation, or the like. In variousembodiments, an in vitro antiviral activity assay may comprise exposingand infecting a cell culture with a known number of infectious viralparticles, for example 200 plaque forming units (pfu). In such anexample, observation of 200 viral plaques following a suitable period ofincubation would be scored as 100% plaque formation. Similarly,observation of 144 viral plaques would be scored as 72% plaqueformation.

In various aspects, a botanical extract or composition may besubsequently combined with an excipient or agent to prepare atherapeutic composition. For example, in various embodiments, a 50:50ratio (vol:wt) of botanical extract composition to VERSABASE gel may beformulated to provide a therapeutic composition. The use of VERSABASEgel in a therapeutic composition of a botanical extract may provide thefollowing benefits: The ammonium acryloyldimethyltaurate/VP Copolymermay act as a gelling agent for the aqueous solution to allow for topicalapplication without dripping or drying out. The aloe vera may enhanceskin penetration of the active botanical constituents allowing fortransdermal uptake. The allantoin may act as a keratolytic agent toimprove moisture binding capacity of the epidermis to also improve drugpenetration into the skin. Other base gels with these activities may beused may be used, but these application-associated activities (includingtransdermal absorption and surface adhesion) are necessary for theefficacy and therapeutic value of the botanical antivirals. Suchalternative gels include: LIPODERM® (Professional Compounding Centers ofAmerica, Inc., Houston, Tex.), PENTRAVAN® (Fargon, Inc., St. Paul,Minn.), and Plutonic Lecithin Organogel. LIPODERM reportedly comprisesethoxydiglycol, water, glycerin, C₁₂-C₁₅ alkyl benzoate, glycerylstearate, dimethicone, cetearyl alcohol, cetearyl glucoside,polyacrylamide, cetyl alcohol, magnesium aluminum silicate, xanthan gum,aloe vera, tocopheryl acetate, bitter almond kernel oil, grape seedextract, wheat germ oil, vitamin-A palmitate, vitamin-C palmitate,ProLipo mutli-emulsion liposomic system, tetrasodium EDTA,phenoxyethanol and sodium hydroxymethyl glycinate. Rather than usingcommercially available bases such as these, ingredients similar to thosefound in these bases may be used to form new bases that can be used inthe therapeutic compositions herein. For example, other fatty alcohols,oils, lipids, gums, polymers, and the like, may be compounded with thebotanical extracts discussed to produce therapeutic compositions withinthe scope of the present disclosure.

In various aspects, the proportion of a botanical extract within acomposition can be adjusted to increase synergistic activity andefficacy.

Methods of Making a Botanical Extract

In accordance with the present disclosure, methods of making a liquidbotanical extract are provided, comprising:

-   -   obtaining plant material from any species within Sarracenia,        Nepenthes, Melissa, Lavandula, Glycyrrhiza, Eleutherococcus,        Hypericum, Darlingtonia, Heliamphora, Roridula, Drosera,        Dionaea, Aldrovanda, Drosophyllum, Triphyophyllum, Catopsis,        Brocchinia, Paepalanthus, Utricularia, Genlisea, Pinguicula,        Ibicella, Byblis, Philcoxia, Stylidium or Cephalotus genera;    -   mixing an extraction solution with the plant material to form a        plant material/extraction solution mixture;    -   storing the mixture for up to about 60-days at room temperature        in amber or clear glass jars or other suitable receptacle,        and/or heating the mixture for up to about 1 day in any suitable        reaction vessel to produce a botanical extract with plant        material suspended therein;    -   removing the plant material from the botanical extract by any        suitable means; and optionally filtering the resulting botanical        extract to remove any remaining particulates.

Fresh plant material may be used for the preparation of liquid botanicalextracts. As used herein, “fresh plant material,” means plants that aresubject to an extraction process less than about 10 days followingharvest. The plant material may comprise any combination of flowers,leaves, stems, shoots, roots, tubers, twigs, buds, and the like, fromany of Sarracenia, Nepenthes, Melissa, Lavandula, Glycyrrhiza,Eleutherococcus, Hypericum, Darlingtonia, Heliamphora, Roridula,Drosera, Dionaea, Aldrovanda, Drosophyllum, Triphyophyllum, Catopsis,Brocchinia, Paepalanthus, Utricularia, Genlisea, Pinguicula, Ibicella,Byblis, Philcoxia, Stylidium or Cephalotus genera.

In accordance with the present disclosure, the extraction solutioncomprises at least one of an alcohol, water, a diol and a polyol. Asused herein, the term “distilled water” may mean water that is purifiedby distillation, including double distillation, and may also be used torefer to water that is purified by other methods, such as reverseosmosis, carbon filtration, micro filtration, ultrafiltration,ultraviolatet oxidation, or electrodialysis. “Alcohol” as used hereinmeans any lower molecular weight C₁-C₁₀ alkanol, such as methanol,ethanol, n-propanol, i-propanol, n-butanol, t-butanol, and the like. Invarious embodiments, 190-proof ethanol is used as the alcohol. Otherforms of ethanol, such as 200-proof ethanol, or any other dilution ofethanol or any alcohol may also be used. “Diol” as used herein means anyhydrocarbon substituted with exactly two-OH (hydroxyl) groups, includingany ether or ester derivative thereof, such as ethylene glycol,propylene glycol, MP diol, ethylene glycol monomethyl ether, propyleneglycol monomethyl ether, and the like. “Polyol” as used herein means anyhydrocarbon substituted with three or more-OH (hydroxyl) groups,including any ether or ester derivative thereof, such as glycerin(glycerol), various sugar alcohols, fatty monoglycerides, fattydiglycerides, fatty triglycerides, and the like. In various embodiments,the extraction solution comprises, for example: ethyl alcohol; water;glycerin; ethyl alcohol, water and glycerin; ethyl alcohol and water;water and glycerin; ethyl alcohol and glycerin; methanol, water andglycerin; methanol and i-propanol; or isopropyl alcohol, water andpropylene glycol monomethyl ether.

In various embodiments, the plant material/extraction solution mixturemay be stored at room temperature for 2-60 days. In further embodiments,the plant material/extraction solution mixture may be boiled/simmeredfor less than one day, such as, for example 30 minutes to 3 or 4 hours.

In various embodiments, the plant material may be removed from thebotanical extract by straining, pressing, and/or by centrifugation.Pressing may be performed using any type of press. The botanical extractmay be optionally filtered through any suitable filtration means toremove various sized particulates remaining after the bulk plantmaterial is removed.

In various embodiments, root, stem and/or leaf material may be used. Forexample, leaves may be separated from plant stems, and then only theleaves used. In other embodiments, entire plants may be used, or onlythe stem material. In further embodiments, roots may be separated fromplants and only the roots used. The plant may be chosen from the groupconsisting of Sarracenia, Nepenthes, Melissa, Lavandula, Glycyrrhiza,Eleutherococcus, Hypericum, Darlingtonia, Heliamphora, Roridula,Drosera, Dionaea, Aldrovanda, Drosophyllum, Triphyophyllum, Catopsis,Brocchinia, Paepalanthus, Utricularia, Genlisea, Pinguicula, Ibicella,Byblis, Philcoxia, Stylidium and Cephalotus genera.

Botanical Extracts

S. purpurea.

In various embodiments, root and/or leaf material may be used to producea Sarracenia botanical extract. For S. purpurea, leaves may beharvested, split longitudinally and any detritus removed. Leaves maythen be subsequently washed and air dried before blending withextraction solution in a blender at a ratio of from about 1:10 to about1:30 grams of plant material:milliliter extraction solution. Forexample, a ratio of about 1:15 grams of plant material:milliliterextraction solution may be used for extraction. In various embodiments,an extraction solution may comprise, for example 190-proofethanol/distilled water/glycerol (63%/32%/5%) or distilledwater/glycerol (25%/75%). In various embodiments, the ethanol may varyfrom about 45% to about 70% and the glycerol may vary from about 0% toabout 10%. In various embodiments, the extraction solution may comprisefrom about 50% to about 80% glycerol, with the remainder being water andno ethanol. The plant material/extraction solution mixture may beincubated at room temperature in amber glass jars for up to about 60days, such as, for example, 7 days. If the extraction solution comprisesonly distilled water, the plant material/extraction solution mixture maybe simmered for about 1-3 hours to perform extraction rather thanincubation at room temperature. Any suitable combination of extractionsolution and incubation condition such as temperature, time, agitation,light protection, and duration may be used in accordance with variousembodiments of the present disclosure.

M. officinalis.

In various embodiments, leaf material from Melissa officinalis may beused to produce a botanical extraction. For example, twelve inch aerialportions of M. officinalis may be harvested, and the leaves may beremoved from stems and the stems discarded. The leaves may besubsequently washed and air dried before blending with extractionsolution at a ratio of from about 1:6 to about 1:20, for example, about1:8, grams plant material milliliter extraction solution, in a blenderin the presence of any suitable extraction solution, such as distilledwater/glycerol (25%/75%). In various embodiments, the percentage ofglycerol in an extraction solution may vary from about 50% to about 80%.The plant material/extraction solution mixture may be incubated at roomtemperature in amber glass jars for from 2-40 days, such as, forexample, 7 days. In accordance with various embodiments, any suitablecombination of extraction solution and incubation condition such astemperature, time, agitation, light protection, and duration may be usedto perform extraction.

L. officinalis.

In various embodiments, leaf material from Lavandula officinalis may beused to produce a botanical extract. For example, six inch floweringstems of Lavandula officinalis may be harvested, and the flowers may beremoved from the stems and the stems discarded. Flowers may be blendedwith extraction solution in a blender at a ratio of from about 1:2 toabout 1:20, for example, about 1:8, grams plant material:miliiliterextraction solution. In various embodiments, extraction solution maycomprise distilled water and glycerol, for example, distilledwater/glycerol (25%/75%). The plant material/extraction solution mixturemay be incubated at room temperature in amber glass jars for from 2-40days, such as, for example, 7 days. Any suitable combination ofextraction solution and incubation condition such as temperature, time,agitation, light protection, and duration may be used in accordance withvarious embodiments of the present disclosure.

H. perforatum.

In various embodiments, leaf and flower material from Hypericumperforatum may be used to produce a botanical extract. For example, sixinch flowering stems of H. perforatum may be harvested, and the flowersand leaves may be removed from stems and stems may be discarded.Flowers/leaves may be blended with extraction solution in a blender at aratio of from about 1:2. to about 1:10, for example, about 1:4, gramsplant material milliliter extraction solution. In various embodiments,extraction solution used for extraction of H. perforatum may compriseethanol, distilled water, and glycerol. For example, 190-proofethanol/distilled water/glycerol (58%/32.%/10%) may be used. In variousembodiments, the percentage of ethanol in an extraction solution used toextract H. perforatum can range from about 40-70%, and the glycerol canrange from about 0% to about 5%. The plant material/extraction solutionmixture may be incubated at room temperature in amber glass jars forfrom 2-40 days, such as, for example, 7 days. Any suitable combinationof extraction solution and incubation condition such as temperature,time, agitation, fight protection, and duration may be used inaccordance with various embodiments of the present disclosure.

E. senticosus.

In various embodiments, fresh plant material or a previously extractedand dried powdered extract of Eleutherococcus senticosus may be used toprepare a botanical extract. In various embodiments, fresh E. senticosusroot material (less than 10 days post harvest) may be blended withextraction solution in a blender at a ratio of from about 1:2 to about1:10 grams plant material:milliliters extraction solution. For example,a ratio of about 1:6 grams plant material:milliliters extractionsolution may be used. In various embodiments, comprising at least one of190 proof ethanol, distilled water and glycerol. For example, E.senticosus root material may be extracted with a mixture of ethanol,distilled water, and glycerol, such as, for example, 42%/53%/5%ethanol-water-glycerol. In various embodiments, the ethanol may comprisefrom about 25% to about 60% of the extraction solution, and the glycerolmay comprise from about 0% to about 5% of the extraction solution. Theliquid/plant mixture may be left at room temperature for 2-40 days, suchas for example for 21 days. For a preparation from a powdered extract,Eleutherococcus senticosus powdered extract (10:1, originally extractedin ethanol and water) may be mixed at a 1:10 ratio with 190-proofethanol/distilled water/glycerol (42%/53%/5%) and agitated daily for 14days. In this case, the ethanol may be present from 25% to about 60% andthe glycerol present at from about 0% to about 5%.

G. glabra.

Either fresh plant material or a previously extracted and dried powderedextract may be used to prepare a liquid extract. Fresh plant material(less than 10 days following harvest) is mixed at a ratio of from 1:2 to1:5 grams plant material to milliliters liquid, with the liquidcomprising at least one of 190 proof ethanol, distilled water andglycerol. F or example, a mixture of 26%/64%/10% ethanol-water-glycerolmay be used as the liquid for extraction. In various embodiments, theethanol may be present from 10% to 50% and the glycerol may be presentfrom about 0% to about 5%. The liquid/plant mixture may be left at roomtemperature for 2-40 days, such as for example for 21 days. For apreparation from a powdered extract, Glycyrrhiza glabra powdered extract(8:1, originally extracted in ethanol and water) may be mixed at a 1:8to 1:10 ratio with a liquid comprising at least one of 190 proofethanol, distilled water and glycerol. For example, a liquid comprising190-proof ethanol/distilled water/glycerol (26%/64%/10%) may be used.The mixture is agitated daily for 2-20 days, such as for example 14days. Any suitable combination of extraction solution and incubationcondition such as temperature, time, agitation, light protection, andduration may be used in accordance with various embodiments of thepresent disclosure.

Other Genera in General:

Other species from each of the six (6) genera represented above(Sarracenia, Melissa, Lavandula, Glycyrrhiza, Eleutherococcus andHypericum) may be used in similar extraction procedures to obtainbotanical extracts. Various other plants may be also be extracted inaccordance with these methods, adapted as necessary for the differentgenera and species therein, including for example, Nepenthes,Darlingtonia, Heliamphora, Roridula, Drosera, Dionaea, Aldrovanda,Drosophyllum, Triphyophyllum, Catopsis, Brocchinia, Paepalanthus,Utricularia, Genlisea, Pinguicula, Ibicella, Byblis, Philcoxia,Stylidium and Cephalotus genera.

Following extraction, a botanical extract, such as any of the botanicalextracts described above or elsewhere herein, may be separated from theplant material used to create the extract, in various embodiments, atincture press may be used to filter the botanical extract using anunbleached paper filter. In various embodiments, a centrifuge may beused to remove the solid debris from an extract. Any suitable means ofseparating liquid extract from solid debris may be used in accordancewith various embodiments of the present disclosure.

Botanical Extract Standardization.

In various aspects, a botanical extract used in botanical extractcomposition may be standardized according to a biological activity, Invarious embodiments, a botanical extract may be standardized withreference to the ability of the extract to inhibit replication of aparticular virus. For example, a botanical extract may be standardizedbased on in vitro antiviral activity against a virus such as such asHSV1, HSV2, VZV, SV-40, or the like. Any suitable target virus againstwhich a formulation comprising a botanical extract of the presentdisclosure may be used can be used in an in vitro antiviral activityassay. Likewise, any suitable in vitro assay that may be used toevaluate the ability of a virus to infect, replicate, or otherwisedemonstrate aspects of a viral disease process may be used for botanicalextract standardization in accordance with the present disclosure.

Standardization of extracts may be performed, for example, on individuallots of plant material, extraction batches, or the like. Suchstandardization may facilitate production of botanical extractcompositions having consistent efficiacy or biological activity despitevariations in factors such as the concentration or quantity of abiologically active constituent in plant biomass obtained from differentsources or extraction process efficiency. In various embodiments,standardization of each botanical extract in a composition comprisingmultiple extracts facilitates the manufacture of a multi-extractcompositions with consistent antiviral activity against a target virusor viruses.

Referring now to FIG. 22, a process 100 for standardizing the biologicalactivity of a botanical extract is provided. In various aspects, abiological activity assay may be prepared 10. Preparation of abiological activity assay 10 may comprise, for example, growing a cellculture to a target cell density and/or preparing a dilution of a viralstock to a target infectious viral partical density. In further aspects,a botanical extract may be prepared 20. Preparation of a botanicalextract 20 may comprise, for example, preparation of an extract from aplant tissue, as described elsewhere herein. Preparation of a botanicalextract may further comprise preparing various concentrations of abotanical extract, such as, for example, a dilution series. In furtheraspects, a process 100 may comprise performing a biological activityassay with the botanical extract 30. In step 30, the variousconcentrations of a botanical extract prepared in step 20 may be used toperform a biological activity assay to assess the activity of theextract. The biological activity of a botanical extract is determinedbased on assay results 40. Following determination of the biologicalactivity of the botanical extract based on the results of a biologicalactivity assay in step 40, the strength of the botanical extract may beadjusted to a target biological activity level in step 50, such as bydilution or concentration of the botanical extract.

In various embodiments, a process 100 for standardizing the biologicalactivity of a botanical extract may comprise a process for standardizingthe antiviral activity of a botanical extract, including one or more ofthe steps of:

-   -   Growing cell cultures to a target cell density for viral        infection;    -   Preparing a dilution of a viral stock to a known plaque forming        unit (pfu) concentration;    -   Preparing a botanical extract;    -   Preparing a serial dilution of the botanical extract to produce        botanical extract treatment concentrations;    -   Treating aliquots of the viral stock with each concentration of        the serial dilution of the botanical extract, along with a        control (no extract) treatment;    -   Adding the treated aliquots of the viral stock to the cultured        cells to produce infected cell cultures,    -   Adding fresh media containing an equivalent botanical extract        concentration to each infected cell culture.    -   Incubating infected/treated cell cultures for an incubation        period.    -   Measuring and recording viral plaque formation.    -   Adjusting the concentration of the botanical extract based on        the determined biological activity.

In various embodiments, preparing a dilution of a viral stock to a knownplaque forming unit concentration involved titrating the viral stock to,for example, 200 pfu per viral stock aliquot. The viral stock aliquotmay then be treated and/or used to infect a cell monoculture. Measuringand recording viral plaque formation may comprise counting the number ofviral plaques and reporting the viral plaque formation as a percentageof plaque formation, wherein the percentage of plaque formation is theobserved number of plaques divided by the expected number of plaquesbased on the titrated viral concentration of the viral stock aliquot.

In various aspects, a botanical extract, botanical extract composition,or a therapeutic composition comprising a botanical extract may bestandardized according to any suitable criteria. For example, in variousembodiments, a botanical extract or a composition thereof may bestandardized based on anti-inflammatory activity. Any suitablebiological activity, quantitative or qualitative chemical marker, or thelike may be used for standardization of a botanical extract or acomposition comprising a botanical extract in accordance with thepresent disclosure.

Botanical Extract Compositions

In various aspects, suitable botanical extracts are provided havinganti-HSV1 activity, including extracts of Sarracenia purpurea, Melissaofficinalis, Lavandula officinalis, Glycyrrhiza glabra, Eleutherococcussenticosus and Hypericum perforatum. These botanical extractsdemonstrate antiviral activity towards HSV1 at comparably low cellcytotoxicity. In further aspects, one or more of these botanicalextracts are combined (e.g., by blending two or more botanical extracts,or compounding them together in a therapeutic composition, etc.) to formbotanical extract compositions.

In various embodiments, botanical extract compositions may comprises twoor more botanical extracts in combination. The combinations may beformulated by various proportions of botanical extracts, such as byvarious volume ratios or by biological activity proportions. Forexample, a botanical extract composition may comprise 50% botanicalextract of Sarracenia purpurea, 15% botanical extract of Melissaofficinalis, 15% botanical extract of Lavandula officinalis, 10%botanical extract of Glycyrrhiza glabra, 5% botanical extract ofEleutherococcus senticosus and 5% botanical extract of Hypericumperforatum. In various embodiments, such as for therapeutic compositionsthat may be used for topical application for herpes virus familyinfections, the botanical extract composition can comprise 50% extractof Sarracenia purpurea, 12% Melissa officinalis, 20% Lavandulaofficinalis, 5% Glycyrrhiza glabra, 8% Eleutherococcus senticosus and 5%Hypericum perforatum, Each botanical extract may be prepared by methodsdescribed in greater detail herein. Any suitable combination ofbotanical extracts in any suitable proportion may be combined to producea botanical extract composition in accordance with various aspects andembodiments.

In various embodiments, botanical extract compositions are set foraccording to standardized anti-HSV1 activity and may comprise any one ormore of:

-   -   a Sarracenia purpurea extract having a standardized anti-HSV1        activity of about 60-300 VIU/ml;    -   a Melissa officinalis extract having a standardized anti-HSV1        activity of about 1000-4000 VIU/ml;    -   a Lavandula officinalis extract having a standardized anti-HSV1        activity of about 75-300 VIU/ml;    -   a Glycyrrhiza glabra extract having a standardized anti-HSV1        activity of about 50-500 VIU/ml;    -   an Eleutherococcus senticosus extract having a standardized        anti-HSV1 activity of about 100-2000 VIU/ml); and    -   a Hypericum perforatum extract having a standardized anti-HSV1        activity adjusted of about 1000-4000 VIU/mL

in various further embodiments, botanical extract compositions are setforth by volume and optionally according to standardized anti-HSV1activity, and may comprise any one or more of:

-   -   about 25-50% v/v Sarracenia purpurea, optionally with a        standardized anti-HSV1 activity adjusted to be from about 60-300        VIU/ml;    -   about 6-12% v/v Melissa officinalis, optionally with a        standardized anti-HSV1 activity adjusted to be from about        1000-4000 VIU/ml;    -   about 10-20% v/v Lavandula officinalis, optionally with a        standardized anti-HSV1 activity adjusted to be from about 75-300        VIU/ml;    -   about 2.5-5% v/v Glycyrrhiza glabra, optionally with a        standardized anti-HSV1 activity adjusted to be from about 50-500        VIU/ml;    -   about 2.5-8% v/v Eleutherococcus senticosus, optionally with a        standardized anti-HSV1 activity adjusted to be from about        100-2000 VIU/ml); and    -   about 4-10% v/v Hypericum perforatum, optionally with a        standardized anti-HSV1 activity adjusted to be from about        1000-4000 VIU/ml

In various further embodiments, botanical extract compositions are setforth according to volume and according to standardized anti-HSV1activity, and may comprise any one or more of:

-   -   about 50% v/v Sarracenia purpurea, optionally with a        standardized anti-HSV1 activity adjusted to about 125 viral        inhibitory units (VIU)/ml;    -   about 12% v/v Melissa officinalis, optionally with a        standardized anti-HSV1 activity adjusted to about 2000 VIU/ml;    -   about 20% v/v Lavandula officinalis, optionally with a        standardized anti-HSV1 activity adjusted to about 150 VIU/ml;    -   about 5% v/v Glycyrrhiza glabra, optionally with a standardized        anti-HSV1 activity adjusted to about 100 VIU/ml;    -   about 8% v/v Eleutherococcus senticosus, optionally with a        standardized anti-HSV1 activity adjusted to about 500 VIU/ml;        and    -   about 10% v/v Hypericum perforatum, optionally with a        standardized anti-HSV1 activity adjusted to about 2000 VIU/ml.

In various further embodiments, botanical extract compositions are setforth according to anti-HSV1 activity and optionally according tovolume.

Human infection with alpha viruses (HSV1, HSV2 and varicella-zostervirus) typically present with lesions on the epidermis. In variousembodiments, a botanical extract composition may be further combinedwith a base gel product to produce a therapeutic composition.Accordingly, in various embodiments, therapeutic compositions areprovided as a suspension of the aqueous extract in a gel to providetopical application and suitable transdermal ‘driving’ capabilities. Infurther embodiments, a gel suspension is prepared using a botanicalextract composition combined with a gel (e.g., VERSABASE). Various otherexcipients may be optionally added, including ammoniumacryloyldimethyltaurate/VP Copolymer, aloe vera, edetate di sodium,allantoin, and methylchloroisothiazolinone/methylisothiazolmone. Invarious embodiments, a botanical extract composition is blended with agel in a proportion suitable for various therapeutic applications. Forexample, in various embodiments, a 50:50 ratio (vol:wt) of a botanicalextract composition to gel (e.g., VERABASE Gel) may be used to produce atherapeutic composition.

In another aspect, a blend for the treatment of cervical dysplasiaassociated with HPV infection is provided. In various embodiments, suchas for therapeutic compositions that may be used for cervical dysplasia,the botanical extract composition can comprise 83.5% extract ofSarracenia purpurea, 7% Melissa officinalis, 1.5% Lavandula officinalis,1.5% Glycyrrhiza glabra, 1.5% Eleutherococcus senticosus and 5%Hypericum perforatum.

In various embodiments, botanical extract compositions are set foraccording to standardized anti-HPV activity and may comprise any one ormore of:

-   -   a Sarracenia purpurea extract having a standardized anti-HPV        activity of about 60-300 VIU/ml;    -   a Melissa officinalis extract having a standardized anti-HPV        activity of about 1000-4000 VIU/ml;    -   a Lavendula officinalis extract having a standardized anti-HPV        activity of about 75-300 VIU/ml;    -   a Glycyrrhiza glabra extract having a standardized anti-HPV        activity of about 50-500 VIU/ml;    -   an Eleutherococcus senticosus extract having a standardized        anti-HPV activity of about 100-2000 VIU/ml; and    -   a Hypericum perforatium extract having a standardized anti-HPV        activity of about 2000 VIU/ml; ranging from 1000-4000 VIU/ml

In various further embodiments, botanical extract compositions are setforth by volume and optionally according to standardized anti-HPVactivity, and may comprise any one or more of:

-   -   about 40-99% v/v Sarracenia, optionally with a standardized        anti-HPV activity adjusted to be from about 60-300 VIU/ml;    -   about 3-14% v/v Melissa officinalis, optionally with a        standardized anti-HPV activity adjusted to be from about        1000-4000 VIU/ml;    -   about 1-4% v/v Lavendula officinalis, optionally with a        standardized anti-HPV activity adjusted to be from about 75-300        VIU/ml;    -   about 1-4% v/v Glycyrrhiza glabra, optionally with a        standardized anti-HPV activity adjusted to be from about 50-500        VIU/ml;    -   about 1-4% v/v Eleutherococcus senticosus, optionally with a        standardized anti-HPV activity adjusted to be from about        100-2000 VIU/ml; and    -   about 2-8% v/v Hypericum perforatium, optionally with a        standardized anti-HPV activity adjusted to be from about        1000-4000 VIU/ml.

In various further embodiments, botanical extract compositions are setforth according to volume and according to standardized anti-HP Vactivity, and may comprise any one or more of:

-   -   about 83% v/v Sarracenia, optionally with a standardized        anti-HPV activity adjusted to about 125 VIU/ml;    -   about 7% v/v Melissa officinalis, optionally with a standardized        anti-HPV activity adjusted to about 2000 VIU/ml;    -   about 2% v/v Lavendula officinalis, optionally with a        standardized anti-HPV activity adjusted to about 150 VIU/ml;    -   about 2% v/v Glycyrrhiza glabra, optionally with a standardized        anti-HPV activity adjusted to be from about 100 VIU/ml;    -   about 2% v/v Eleutherococcus senticosus, optionally with a        standardized anti-HPV activity adjusted to be from about 500        VIU/ml; and    -   about 4% v/v Hypericum perforatum, optionally with a        standardized anti-HPV activity adjusted to be from about 2000        VIU/ml.

In various further embodiments, botanical extract compositions are setforth according to anti-HPV activity and optionally according to volume.

Botanical extract compositions wherein one or more botanical extractsare added, substituted, or deleted may be produced and/or formulatedinto a therapeutic composition in accordance with various embodiments ofthe present disclosure. Such modifications of the botanical extractcompositions of the present disclosure may be produced for any suitablepurpose, such as, for example, to modulate a synergistic activity orefficacy of a composition.

In various embodiments, a botanical extract composition can be comprisedentirely of an extract of Sarracenia purpurea.

In accordance with various embodiments, the relative proportions of eachbotanical extract in a mixture can vary. In various embodiments, abotanical extract composition can comprise 40-99% extract of Sarraceniapurpurea, 1-30% Melissa officinalis, 1-30% Lavandula officinalis, 1-30%Glycyrrhiza glabra, 1-20% Eleutherococcus senticosus and 1-20% Hypericumperforatum.

In various embodiments, the botanical extracts from species includingSarracenia purpurea, Melissa officinalis, Lavandula officinalis,Glycyrrhiza glabra, Eleutherococcus senticosus and/or Hypericumperforatum are formulated into a blend to provide optimal activity.

In various embodiments, a botanical extract composition may comprise anysuitable combination of botanical extracts at any individual volumeproportion relative to the total volume of the composition. Likewise, abotanical extract having have any suitable standardized antiviralactivity level (expressed in VIU), determined for any viral agent, maybe used to produce a botanical extract composition. In variousembodiments, the volume proportion of a botanical extract in acomposition may be adjusted based on a measured antiviral activity levelof the extract. Similarly, the antiviral activity of an extract per unitvolume may be adjusted, for example, by dilution or concentration, toprovide a standardized antiviral activity level suitable for adding theextract to a botanical extract composition at a volume proportioncompatible with the volumes or activities of other botanical extractsthat may be present in the composition. The foregoing embodiments arepresented by way of example, and not by way of limitation. Any suitableproportion of an individual botanical extract in a botanical extractcomposition, with the individual botanical extract having any suitablerange of antiviral or other standardized biological activity, is withinthe scope of the present disclosure. In various embodiments, one or moremechanisms of action may be associated with various botanical extractsor botanical extract compositions.

With reference to FIG. 4, various the viral targets are shown. FIG. 4illustrate a possible mechanism of action of a poxvirus therapeuticcomposition in accordance with various embodiments of the presentdisclosure. The illustration indicates the general replication cycle ofVACV. Cidofovir (VISTIDE®, Gilead Sciences Inc., Foster City, Calif.) isknown to interfere with genome replication, while ST-246 is known tointerfere with the maturation step.

While not desiring to be bound by theory, it is believed that S.purpurea inhibits the replication of poxviruses (including vacciniavirus, monkeypox virus and variola virus) soon after entry of the virusinto the cell by inhibiting viral mRNA synthesis, such as between theviral entry and early transcription steps illustrated in FIG. 4. Uponscreening of the spectrum of antiviral activity that may be associatedwith S. purpurea, the extract was found to have antiviral activityagainst other non-related DNA viruses including HSV1 and SV40 (apapovavirus related to human papillomavirus), but not toward RNA virusesincluding mouse hepatitis virus, reovirus, vesicular stomatitis virus,and encephalomyocarditis virus. Of the botanicals included withinvarious botanical extract compositions described herein, such as theblend of all Species II Extracts, S. purpurea has been one of the morewell characterized. When cells are infected with HSV1 typical cytopathiceffects are observed. If, following infection, the cells are treatedwith S. purpurea extract, the cytopathic effect could be virtuallyeliminated. In order to assess at which point in the virus replicationcycle S. purpurea was acting on the HSV1, a Western blot was performedto detect the level of an immediate-early protein synthesized by thevirus (ICP4) (FIG. 7).

Following treatment with S. purpurea, the accumulation of ICP4 is almostnon-detectable. This suggests that the S. purpurea is targeting an earlyevent in the virus replication cycle, either virus uptake into the cell,immediate-early transcription, or immediate-early protein synthesis.Given the previous results with poxviruses, it is believed the extractis likely targeting viral transcription.

As further described herein, the antipoxvirus activity associated withS. purpurea extracts was found not to be limited to a particular genusof carnivorous botanicals. Extracts prepared from other carnivorousgenera also demonstrated antiviral activity. Such carnivorous generaincluded Nepenthes, Drosera, Dionaea, Darlingtonia, and Pinguicula.

A number of other plant genera were assayed for conservation of theantiviral activity associated with S. purpurea. Species from othergenera tested, including Sarracenia spp., Nepenthes spp., Drosera spp.,Dionaea spp., Darlingtonia spp., and Pinguicula spp., all have antiviralactivity against vaccinia virus. The strongest antiviral activity hasbeen associated with Sarracenia spp. and Nepenthes spp. For Sarraceniaspp. and Nepenthes spp., this antiviral activity has been observedtoward poxviruses, herpesviruses and papovaviruses.

The genus Nepenthes includes a carnivorous pitcher plant. Nepenthes issimilar to Sarracenia in terms of general appearance, but isgeographically distributed in very different regions of the worldcompared to Sarracenia. For other species, this conserved antiviralactivity has only been tested against vaccinia virus, although it may beeffective against herpes and papilloma/polyomaviruses.

Our screening of botanicals for activity against HSV1 ranked M.officinalis as one of the strongest anti-HSV1 extracts of the variousbotanical extracts disclosed herein.

Current data is limited on the mechanism of action of the various otherbotanical extracts within the Species II Extracts. For Lavandulaofficinalis, preliminary data suggests the anti-herpes viral target islikely an early event, possibly affecting viral entry into the cell. ForGlycyrrhiza glabra, treatment of HSV1 infected cells results in theproduction of a smaller plaque phenotype. This is a very differenteffect compared to Melissa extracts, where plaque numbers are reducedwhile plaque size remains consistent. This may suggest that Glycyrrhizaextracts may be affecting a later event in the viral replication cycleor viral cell-to-cell spread or altering the phenotype of subsequentlyinfected cells (i.e. inducing a cellular antiviral response orrepressing factors/receptors required by the virus). For Eleutherococcussenticosus, the antiviral target appears to be an early event in theviral replication cycle, but likely unique from Melissa or Lavandulasince this botanical does not affect EHV-1 replication whereas bothLavandula and Melissa can inhibit replication of EHV-1. For Hypericumperforatum, a mechanism of action against HSV1 has not yet beenidentified.

Since many of these botanicals target different points in thereplication cycle, various combinations of the Species II Extractsprovide synergistic activity towards the inhibition of viralreplication. Blends that comprise Species II Extracts may producemulti-targeted efficacy against viral replication, wherein the differentbotanicals inhibit different targets in the viral replication cycle,thereby effectively inhibiting viral replication with strongerefficiency. In addition, given that these botanical extracts inhibit thevirus at different sites, it is assumed that the development ofpotential viral resistance to such a blended therapy will be greatlyreduced.

Methods of Preparing Therapeutic Compositions Comprising BotanicalExtracts

Methods for the preparation of therapeutic compositions in accordancewith the present disclosure comprise formulating one or more of thebotanical extracts disclosed herein with one or more pharmaceuticallyacceptable excipients or carriers to form a solid, semi-solid or liquid.Solid compositions include, but are not limited to, powders, tablets,dispersible granules, capsules, cachets, and suppositories. Liquidformulations include solutions a botanical extract is present, such asemulsions or a solution containing liposomes, micelles, or nanoparticlescomprising a botanical extract as disclosed herein. Semi-solidformulations include, but are not limited to, gels, suspensions andcreams. The form of the therapeutic compositions described hereininclude liquid solutions or suspensions, solid forms suitable forsolution or suspension in a liquid prior to use, or as emulsions. Theseformulations also optionally contain minor amounts of nontoxic,auxiliary substances, such as wetting or emulsifying agents, pHbuffering agents, and so forth In various embodiments, a botanicalextract, or a blend of botanical extracts, may be compounded into atherapeutic composition with one or more pharmaceutically acceptableexcipients. For example, for dermal or epithelial topical therapeuticuse of a single botanical extract or a blend of extracts, the aqueousmixture is suspended in a transdermal base or other pharmaceutical base,such as a gel, to provide such drug delivery properties as adherence andtransdermal ‘driving’ capabilities, for example.

Pharmaceutically acceptable excipients include for example, solvents(such as water, glycols, alcohols, ethers), and solubilizers andcarriers (such as fatty acids, glycerin, monoglycerides, diglycerides,triglycerides, sorbitan fatty esters, alkoxylated fatty acids,alkoxylated fatty acid esters, lecithin, and the like). Pharmaceuticallyacceptable excipients have been amply described in a variety ofpublications, including, for example, A. Gennaro (2000) “Remington: TheScience and Practice of Pharmacy,” 20th edition, Lippincott, Williams, &Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999)PI. C. Ansel et al., eds., T.sup.th ed., Lippincott, Williams, &Wilkins; and Handbook of Pharmaceutical Excipients (2.000) A. H. Kibbeet al., eds., 3.sup.rd ed. Amer. Pharmaceutical Assoc., all of which areherein incorporated by reference.

Pharmaceutically acceptable excipients, such as emulsifiers,solubilizers, emollients, vehicles, adjuvants, carriers or diluents, arecommercially available. Moreover, pharmaceutically acceptable auxiliarysubstances, such as pH adjusting and buffering agents, tonicityadjusting agents, stabilizers, wetting agents, preservatives and thelike, are commercially available.

In various embodiments, the botanical extracts of the present disclosurecan be administered topically. The botanical extracts described hereincan be formulated into a variety of topically administrableformulations, such as solutions, suspensions, lotions, gels, pastes,medicated sticks, balms, creams or ointments. Such therapeuticcompositions optionally contain solubilizers, stabilizers, tonicityenhancing agents, buffers and preservatives. For topical administration,the present botanical extracts may be applied in liquid form. However,it will generally be desirable to administer them to the skin asformulations, in combination with a dermatologically acceptablecarrier/gel, which may be a solid or a liquid. The resultant liquid orgel therapeutic compositions can be applied from absorbent pads, used toimpregnate bandages and other dressings, used in transdermal patches, orsprayed onto the affected area using pump-type or aerosol sprayers.

Examples of useful dermatological formulations which can be used todeliver the botanical extracts may be found in, for example Jacquet etal. U.S. Pat. No. 4,608,392; Geria U.S. Pat. No. 4,992,478; Smith et al.U.S. Pat. No. 4,559,157 and Wortzman U.S. Pat. No. 4,820,508, each ofwhich is incorporated in its entirety herein by reference.

Various embodiments provide a therapeutic composition comprising abotanical extract described herein and a gel, or other suitable basecomposition, providing adherence and transdermal driving capabilities(absorption through the skin). In various embodiments, the therapeuticcomposition comprises 25% of a botanical extract and 75% of at least onepharmaceutical excipient such as a base gel composition. The therapeuticcomposition can range from 25-70% of a botanical extract andcorresponding 75-30% pharmaceutical excipient(s).

In various embodiments, such as for formulations used for topicalapplication for herpes virus family infections, a therapeuticcomposition may comprise 50% of a botanical extract composition and 50%gel. In various embodiments, such as for formulations used for cervicaldysplasia applications, a therapeutic composition may comprise 60% of abotanical extract composition and 40% gel. The botanical extractcomposition can range from 25-70% of a botanical extract described aboveand corresponding 75-30% gel.

In various embodiments, the compounding base is a transdermal drivergel, such as, for example, VERSABASE gel. VERSABASE gel is manufacturedby Professional Compounding Center of America and reportedly containsammonium acryloyldimethyltaurate/VP Copolymer, aloe vera, edetatedisodium, allantoin, methylchloroisothiazolinone andmethylisothiazolinone.

In various embodiments, it is believed the VERSABASE gel provides thefollowing: ammonium acryloyldimethyltaurate/VP Copolymer acting as agelling agent for the aqueous solution to allow for topical applicationwithout dripping or drying out; aloe vera acting to enhance skinpenetration of the active botanical constituents allowing fortransdermal uptake; allantoin acting as a keratolytic agent to improvemoisture binding capacity of the epidermis to also improve drugpenetration into the skin.

In certain embodiments, other commercially available compounding basesmay be used. Such alternative bases include but are not limited toLIPODERM, PENTRAVAN, and Pluronic Lecithin Organogel (PLO). PLO base isa thermoreversible gel that is liquid at low temperatures (4° C.) andbecomes more solid as the temperature increases (body temperature). Thisproperty makes it useful for applications, especially on the cervix orvaginal tract of patient, for transdermal drug delivery.

Alternatively, individual pharmaceutically acceptable ingredients, suchas fatty acids, fatty acid esters, alkoxylated fatty acids, alkoxylatedfatty acid esters, monoglycerides, diglycerides, triglycerides, sorbitanfatty acid esters, and the like, can be blended in any combination withthe various botanical extracts or blends of extracts as desired toproduce therapeutic compositions within the scope of the presentdisclosure. More particularly, the botanical extracts of the presentdisclosure can be compounded into therapeutic compositions bycombination with appropriate, pharmaceutically acceptable carriers ordiluents, and may be formulated into preparations in solid, semi-solid,liquid or gaseous forms, such as tablets, capsules, powders, granules,ointments, creams, mulls, gels, solutions, suppositories, injections,inhalants and aerosols.

In various embodiments, pharmaceutical excipients include fatty acids.Any fatty acid, or combination of fatty acids, may be blended with abotanical extract or blend of extracts herein to produce a therapeuticcomposition. For example, any C₆-C₂₄ fatty acid may be used in thepharmaceutical formulas of the present disclosure. Fatty acids for useherein also include C₆-C₂₄ fatty acids having any degree of unsaturationin the carbon chain. Fatty acids for use herein may be from natural oiland fat sources such as tallow, lard, coconut oil, palm oil, peanut oil,rice bran oil, olive oil, cottonseed oil, wheat germ oil, soy bean oil,corn oil, sunflower oil, and safflower oil, amongst others. Natural oilsmay supply a distribution of fatty acid chain lengths. For therapeuticcompositions within the scope of the present disclosure, any combinationof free fatty acid, natural oils, hydrogenated oils, partiallyhydrogenated oils, vegetable and animal fats, monoglycerides,diglycerides and triglycerides may be used, and may be combined with anyother emulsifiers, emollients, carriers, solubilizers, solvents, and thelike, as desired.

In various embodiments, a therapeutic composition comprises a botanicalextract and a fatty acid. For example, a therapeutic composition withinthe scope of the present disclosure comprises a botanical extract and afatty acid substance. For example, the plant species Sarracenia purpureamay be used. In various embodiments, a therapeutic composition comprisesany botanical extract or blend of extracts discussed above combined withat least one fatty acid.

In various embodiments, administration of a botanical extract ortherapeutic composition thereof can be achieved in various ways,including preparations for oral, buccal, rectal, parenteral,intraperitoneal, intradermal, subcutaneous, intramuscular, transdermal,intratracheal, etc., administration.

In various embodiments, botanical extracts of the present disclosure areformulated for oral administration by combining the botanical extract(s)with, e.g., pharmaceutically acceptable carriers or excipients. Invarious embodiments, the compounds of the present disclosure areformulated in oral dosage forms that include, for example, tablets,powders, pills, capsules, liquids, gels, syrups, elixirs, slurries,suspensions, and the like.

For oral preparations, the botanical extracts can be combined withappropriate additives to make liquids, tablets, powders, granules orcapsules, for example, with conventional additives, such as lactose,mannitol, corn starch or potato starch; with binders, such ascrystalline cellulose, cellulose derivatives, acacia, corn starch orgelatins; with disintegrators, such as corn starch, potato starch orsodium carboxymethylcellulose; with lubricants, such as talc ormagnesium stearate; and if desired, with diluents, buffering agents,moistening agents, preservatives and flavoring agents.

In various embodiments, therapeutic compositions for oral use areobtained by mixing one or more solid excipients with one or more of thebotanical extracts described herein, optionally grinding the resultingmixture, and processing the mixture of granules, after adding suitableauxiliaries, if desired, to obtain tablets. Suitable excipients includefillers such as sugars, (e.g. lactose, sucrose, mannitol, or sorbitol);cellulosic substances: (e.g., maize starch, wheat starch, rice starch,potato starch, gelatin, gum tragacanth, methylcellulose,microcrystalline cellulose, hydroxypropylmethylcellulose, sodiumcarboxymethylcellulose); or other materials such as, for example,polyvinylpyrrolidone (PVP or “povidone”) or calcium phosphate. Inspecific embodiments, disintegrants can be added. Disintegrants include,for example, cross-linked croscarmellose sodium, polyvinylpyrrolidone,agar, or alginic acid and sodium alginate.

In various embodiments, tablets can be provided with one or moresuitable coatings. In specific embodiments, concentrated sugar solutionsare used for coating the dosage form. Sugar solutions can optionallycontain additional components, such as for example, gum arable, talc,polyvinylpyrrolidone, carbopol polyacrylate gels, polyethylene glycol,titanium dioxide, lacquer solutions, organic solvents or solventmixtures. Colorants can be added to the coatings for marketing or doseidentification, or other purpose.

In various embodiments, therapeutically effective amounts of at leastone of the botanical extracts of the present disclosure are formulatedinto other oral dosage forms. Oral dosage forms include push-fitcapsules made of gelatin, as well as soft sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. In variousembodiments, push-fit capsules contain the botanical extract(s) mixedwith one or more fillers. Fillers include, for example, lactose, binderssuch as starches, and/or lubricants such as talc or magnesium stearateand, optionally, stabilizers. In various other embodiments, softcapsules can contain one or more botanical extracts dissolved orsuspended in a suitable liquid. Suitable liquids include, for example,one or more fatty oils, glycerin, glycerides, liquid paraffin, orvarious polyethylene glycols.

In various embodiments, therapeutically effective amounts of at leastone of the botanical extracts of the present disclosure are formulatedfor buccal or sublingual administration. Formulations suitable forbuccal or sublingual administration include, for example, tablets,lozenges, or gels. In still other embodiments, the botanical extractsdescribed herein are formulated for parental injection, includingformulations suitable for bolus injection or continuous infusion. Inspecific embodiments, formulations for injection are presented in unitdosage form (e.g., in ampoules) or in multi-dose containers.

In various embodiments, the botanical extracts can be formulated intoformulations for injection by dissolving, suspending or emulsifying oneor more botanical extracts or in an aqueous or nonaqueous solvent, suchas vegetable or other similar oils, synthetic aliphatic acid glycerides,esters of higher aliphatic acids or propylene glycol: and if desired,with conventional additives such as solubilizers, isotonic agents,suspending agents, emulsifying agents, stabilizers and preservatives.Parenteral injection formulations optionally contain excipients such assuspending, stabilizing and/or dispersing agents. In specificembodiments, therapeutic compositions for parenteral administrationinclude aqueous solutions of the botanical extracts in water-solubleform. In additional embodiments, suspensions of the botanical extractsherein are prepared as oily injection suspensions. Suitable lipophilicsolvents or vehicles for use in the therapeutic compositions describedherein include, for example, fatty acid blends such as those found innatural oils such as peanut oil, fatty acid esters, mono-, di- andtriglycerides, or liposomes. In various embodiments, aqueous injectionsuspensions contain substances which increase the viscosity of thesuspension, such as sodium CMC, sorbitol or dextran. Optionally, thesuspension can include suitable stabilizers or other agents thatincrease the solubility of the botanical extracts to allow for highlyconcentrated solutions. Alternatively, the botanical extract can bedehydrated or lyophilized into a powdered form for later mixing with asuitable vehicle by the practitioner.

In various embodiments, the botanical extracts of the present disclosurecan be formulated for administration by inhalation. Inhalationadministration can include an intranasal spray. Various forms suitablefor administration by inhalation include aerosols, mists or powders.Therapeutic composition comprising botanical extracts of the presentdisclosure can be conveniently delivered in the form of an aerosol spraypresentation from pressurized packaging or a nebulizer, e.g. with theuse of a propellant (e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or thelike). In various embodiments, the dosage unit of a pressurized aerosolis determined by selection of a valve that can meter the dose. Incertain embodiments, capsules and cartridges of the formulation can beprovided for use within a pressurized delivery system. In variousembodiments, the botanical extracts of the present disclosure can beformulated into liquid formulations sprayable from non-aerosolpackaging.

In various embodiments, the botanical extracts of the present disclosurecan be formulated into rectal compositions such as enemas, rectal gels,rectal foams, rectal aerosols, suppositories, jelly suppositories, orretention enemas, which contain suppository bases such as cocoa butteror other glycerides, in addition to synthetic polymers such aspolyvinylpyrrolidone, PEG, and the like. Suppository forms can include alow-melting wax such as, but not limited to, a mixture of fatty acidglycerides. Furthermore, the botanical extracts can be formulated intosuppositories by mixing with a variety of bases such as emulsifyingbases or wafer-soluble bases. The suppository can include vehicles suchas cocoa butter, carbowaxes and polyethylene glycols, which melt at bodytemperature, yet are solidified at room temperature.

One of skill in the art will readily appreciate that dose levels canvary as a function of the specific compound, the severity of thesymptoms and the susceptibility of the subject to side effects. Theamount of the botanical extracts required for use in treatment will varynot only with the particular botanical extracts and finishedpharmaceutical formulations selected, but also with the route ofadministration, the nature of the condition being treated and the ageand condition of the patient and will be ultimately at the discretion ofthe attendant physician or clinician.

In various embodiments, botanical extracts and therapeutic compositionsthereof, used in the methods described herein, may be delivered by acontinuous delivery system. The term “continuous delivery system” isused interchangeably herein with “controlled delivery system” andencompasses continuous (e.g., controlled) delivery devices (e.g., pumps)in combination with catheters, injection devices, and the like, a widevariety of which are known in the art.

The dosage forms for oral or rectal administration such as syrups,elixirs, and suspensions may be provided wherein each dosage unit, forexample, teaspoonful, tablespoonful, tablet or suppository, contains apredetermined amount of the botanical extract. Similarly, dosage formsfor injection or intravenous administration may comprise the therapeuticcomposition containing a botanical extract as a solution in sterilewater, normal saline or another pharmaceutically acceptable carrier.

The term “unit dosage form,” as used herein, refers to physicallydiscrete units suitable as unitary dosages for human and animalsubjects, each unit containing a predetermined quantity of botanicalextract of the present disclosure calculated in an amount sufficient toproduce the desired effect in association with a pharmaceuticallyacceptable diluent, carrier or vehicle. The specifications for the novelunit dosage forms of the present disclosure depend on the particularbotanical extract employed and the effect to be achieved, and thepharmacodynamics associated with each botanical extract in the host.

In various embodiments, combination therapy comprises the concurrentadministration of at least one dose of a therapeutic compositioncomprising a botanical extract and an additional antiviral formulationeffective against a virus or a cancer. As used herein, the term“concurrently” means that the two formulations are administeredseparately and are administered within about 5 seconds to about 15seconds, within about 15 seconds to about 30 seconds, within about 30seconds to about 60 seconds, within about 1 minute to about 5 minutes,within about 5 minutes to about 15 minutes, within about 15 minutes toabout 30 minutes, within about 30 minutes to about 60 minutes, withinabout 1 hour to about 2 hours, within about 2 hours to about 6 hours,within about 6 hours to about 12 hours, within about 12 hours to about2.4 hours, or within about 2.4 hours to about 48-hours of one another.As used herein, the term “simultaneously” means that the twoformulations are administered at the same time.

Useful dosages of the botanical extracts herein can be determined bycomparing their in vitro and in vivo activity in animal models. Methodsfor the extrapolation of effective dosages in mice, and other animals,to humans are known to the art; for example, see U.S. Pat. No.4,938,949, incorporated in its entirety herein by reference.

Given below are the following non-limiting Examples of variousembodiments:

Anti-Poxvirus Activity

Initial analysis was done to measure the ability of S. purpurea extractto inhibit poxvirus infections in vitro. We examined the ability of S.purpurea to prevent vaccinia virus (VACV) plaque formation in cellstreated immediately after infection with increasing amounts of extract,administered once or every six hours, for a total of 24 hours. Referringnow to FIG. 1A, a dose dependent reduction in VACV plaquing efficiencyassociated with increasing amounts of S. purpurea extract was observed.RK-13 cells were infected with 150 plaque forming units (“pfu”) of VACVfollowed by the addition of the indicated concentration of S. purpureaextract to the cell culture media. Cells were treated one-time only withthe extract (black bars) or every 6 hours with fresh extract (graybars). After 48 hours, plaques were visualized and quantified. Errorbars represent standard deviation (n=3). To ensure S. purpurea wasresponsible for the decrease in plaquing efficiency, cells were treatedwith 45%:50%:5% ethanol:distilled water:glycerol (carrier for the S.purpurea extract) prior to infection. This carrier treatment did notaffect VACV plaquing efficiency. We subsequently assayed for the abilityof VACV to replicate in cells under single-cycle conditions, treatedonce or every six hours, with S. purpurea extract. Treatment with S.purpurea began immediately following VACV infection and viral titerswere determined every six hours. Referring now to FIG. 1 B, S. purpureatreatment resulted in a dramatic decrease in VACV replication, whencompared to the untreated cells. RK-13 cells were infected with VACV atan multiplicity of infection (“MOI”)=10 followed by addition ofethanol/glycerol carrier (closed diamonds) or the addition of 2.5 μl S.purpurea extract/ml media (open squares and closed triangles). Cellswere treated one-time only with the extract (open squares) or every 6hours with fresh extract (closed diamonds). Cells were harvested at theindicated times and viral titers determined. Error bars representdeviation between assays (n=2).

A single treatment of S. purpurea, caused a 100-1000 fold reduction inVACV replication throughout the course of the infection, however someviral replication was still observed. In cells treated with freshextract every six hours, a 10,000-fold decrease in VACV replication wasobserved. Multiple treatments with S. purpurea completely abolished VACVreplication since titers did not increase over the course of theinfection. In cells treated with the carrier, VACV replicated to levelssimilar to that seen in untreated cells (data not shown). To furtherdetermine the efficacy of using S. purpurea to treat a poxvirusinfection, the selectivity index (SI) associated with the extract wasdetermined. Referring now to FIG. 1C, for viral translation levels(closed squares), HeLa cells were infected with VACV at an MOI=10followed by the addition of the indicated concentrations of S. purpureaextract/ml media. At 6 hours post infection (“HPI”), cell lysates wereprepared, the VACV E3L protein detected by Western blot, and quantified.For cell viability, HeLa cells were treated with the indicatedconcentrations of S. purpurea extract (closed squares) orethanol/glycerol carrier (closed triangles) for 6 hours and the numberof viable cells determined by a trypan blue exclusion assay. In S.purpurea treated cells, the EC50 required to inhibit VACV replicationwas 10-15 μL/mL, while the CC50 was 70-75 μL/mL of the extract, whichresults in a SI of approximately 5-7 (FIG. 1C). This SI is similar tothat reported for cidofovir (CDV), an already proven treatment forpoxvirus infections. Additionally, a 25 μL/mL sufficiently-inhibitedviral protein translation to a level that prevented VACV replication(FIG. 1B) without inducing cellular cytotoxicity (FIG. 1C).

In various embodiments, the antipoxvirus activity associated with S.purpurea extracts was found not to be limited to Sarracenia. Extractsprepared from other genera also demonstrated antiviral activity. Suchgenera included Nepenthes, Drosera, Dionaea, Darlingtonia andPinguicula.

Anti-Poxvirus Activity-Mechanism of Action

In order to understand the mechanism of action associated with S.purpurea, various treatment schedules were tested. Referring now toFIGS. 2A and 2B, HeLa cells were infected with VACV at an MOI=10followed by the addition of 25 μl S. purpurea extract/ml mediaimmediately (0 mm) or at 15, 30, 60 or 120 min post infection. ‘Notreatment’ cells received ethanol/glycerol carrier only. For FIG. 2A, at6 HPI, the cell monolayers were photographed. For FIG. 2B, at 3 HPI,cell lysates were prepared and the VACV E3L protein or total VACVproteins detected by Western blot. The asterisks (*) in the figuresindicate the position of VACV proteins and the VACV-E3L protein.Duplicate experiments were performed at 6 HPI (not shown) Referring nowto FIG. 2C, Hela cells were infected with a VACV construct expressingcyan fluorescent protein fused to the viral A5 core protein. In thefirst panel, the infection was maintained at 4° C. For the middle andlast panel, the infection was done at 37° C., in the absence or presenceof S. purpurea, respectively. With reference now to FIG. 2D, HeLa cellswere mock infected or infected with VACV at an MOI=10 followed by theaddition of 2.5 ul S. purpurea extract/ml media or ethanol/glycerolcarrier. At 4 HPI, the cell monolayers were radiolabeled with[³⁵S]-methionine, cell lysates prepared, proteins separated by SDS-PAGE,and visualized by autoradiography. The asterisks (*) indicate theposition of VACV proteins.

In cells treated with a single dose of S. purpurea overnight prior toinfection with VACV followed by washing, no inhibition of VACVreplication was observed, suggesting the extract does not induce acellular antiviral component. Moreover, treating a purified VACV stockwith S. purpurea did not affect replication of the virus, suggestingthat the extract does not have a direct effect on free virus particles.

Further, activity against varicella-zoster virus (VZV) was assessed.Again, infection of cells in culture with VZV leads to a distinctcytopathic effect. Treatment of infected cells with S. purpurea extractimmediately after infection was able to completely abolish thiscytopathic effect (FIG. 9). Notably, these assays were performed with asingle dose of S. purpurea extract (on day 1) and cytopathic effectsmeasured on days 7 and 9. This inhibition in the replication of VZV byS. purpurea was confirmed by Western blot to detect the g1 protein ofthe virus. From this assay, this protein could not be detected in cellsinfected by VZV which were treated with S. purpurea extract. Theseresults suggest that the S. purpurea extract can effectively inhibit thereplication of alpha herpes virus members including HSV1, VZV, HSV2 andEHV1.

Referring now to FIGS. 32A-32E, related Sarracenia species were testedfor similar anti-herpes virus activity. S. elata (FIG. 32B), S. flava(FIG. 32C), S. leucophylla (FIG. 32D) and S. rubra (FIG. 32E) alldemonstrated similar antiviral activity against HSV 1 compared to S.purpurea (FIG. 32A). For this study, Vero cells were infected with 100pfu HSV1 followed by the addition of increasing concentrations of theindicated botanical extracts prepared from various Sarracenia species(per ml media). At 4 days post infection (“DPI”), cells were stainedwith crystal violet and plaques counted. These results suggest thatmultiple (if not all) species of Sarracenia encode components to inhibitthe replication of alpha herpes viruses. M. officinalis mechanism ofaction

Referring now to FIG. 34, when cells were infected with HSV1, HSV2. orEHV1, typical cytopathic effects were observed. For this study, Verocells were infected with HSV1, HSV2, or EHV1 at an MOI=10. Infectedcells were treated with increasing concentrations of M. officinalisextract. At 3-4 DPI, viral CPE was visualized by microscopy andphotographed. If following infection, the cells were treated with M.officinalis extract, this cytopathic effect could be inhibited in anHSV1 and HSV2 infection and reduced in an EHV1 infection. To assesswhether extracts of M. officinalis could inhibit viral spread fromcell-to-cell, Vero cell monolayers were infected with 100 plaque formingunits of HSV1. Three hours later, any remaining viral inoculum wasremoved and the cells were treated with increasing concentrations of M.officinalis. Viral cytopathic effect was monitored and when full CPE wasobserved in the untreated dish, the cell monolayers were stained withcrystal violet. As shown in FIG. 35A, M. officinalis was able to preventthe spread of HSV1 from infected cells to non-infected ones. For thisstudy, Vero cells were infected with 100 pfu HSV1. At 4 HPI, the cellswere treated with the indicated concentrations of M. officinalisextract. As a control, a comparable set of plates were treated withhuman immunoglobulin (FIG. 35B). The virus infection was allowed toprogress until complete CPE in the untreated plate (6 days). At thattime, cells were stained with crystal violet and viral plaque/CPErecorded. As a control, cells were treated with human immunoglobulinwhich has previously been shown to inhibit HSV1 cell-to-cell spread.

Referring now to FIG. 36, in order to assess at which point in the virusreplication cycle M. officinalis was acting on the HSV1, cells wereinfected with HSV1 and treated with M. officinalis at multiple timespost infection. For this study, Vero cells were infected with HSV1 at anMOI=10. Cells were treated with M. officinalis extract at the indicatedtimes post infection. At 24 HPI, cells lysates were prepared and thelevels of HSV1 ICP4, ICP8 and gD determined by Western Blot analysis.Antiserum to GAPDH was used as a control. Viral replication wasmonitored by Western blot of an immediate-early viral protein (ICP4), anearly viral protein (ICP8) and a late viral protein (gD). M. officinalistreatment must occur either prior to infection or during infection toinhibit HSV1 replication (FIG. 36). This suggests that the M.officinalis is targeting an early event in the virus replication cycle,likely either directly killing the virus, virus attachment to the cell,or virus uptake into the cell. In order to determine the exact target ofanti-herpes activity associated with M. officinalis, multipleexperiments were performed to measure the effect of the extract on thefree virus (direct virucidal), viral attachment to the cell, or viralentry into the cell.

With reference now to FIGS. 37A-37C, the inhibitory effects of M.officinalis extract on HSV1 attachment to the cell are illustrated. Forthis study, standard viral entry (FIG. 37A), viral attachment (FIG. 37B)and direct virucidal assays (FIG. 37C) were performed, the results ofwhich are illustrated. For the standard viral entry assay, virus wasincubated with cells for 3 hrs at 4° C., followed by a wash. Virusexposed cells were treated with different concentrations of extract inmedium for 2 hrs at 37° C. Free virus was inactivated and washed fromthe cells. Cells were stained at 3 DPI. For the direct virucidal assay,virus was treated with a botanical extract at the indicatedconcentrations for four different incubation times ranging from 0 to 120min. The virus was then pelleted, resuspended, and viral activitytitered. There was an observable effect on viral entry and a directvirucidal effect, but these required at least a 10 to 30-fold higherdose of extract, respectively. Therefore, these data support that M.officinalis inhibits HSV1 by blocking viral attachment to the cell. Forthe viral attachment assay, virus was incubated was cells as for theviral entry assay, except botanical extract was also added at theindicated concentrations during this step. Cells were washed, andstained at 3 DPI. HSV1 attachment to a cell requires binding toheparin-sulfate. An in vitro experiment was performed to determine if M.officinalis could block HSV1 binding to heparin attached to an agarosebead. In this assay, HSV1 was incubated with heparin-agarose in thepresence or absence of M. officinalis or free heparin. Virus binding tothe bead was detected by Western blot to the viral gD protein.

Referring now to FIG. 38, the M. officinalis extract was able to blockHSV1 binding to heparin-agarose. For this study, 10 million HSV1particles were incubated with heparin-agarose. As indicated, excesssoluble heparin or M. officinalis extract was added during the bindingassay. Following incubation for 1 hour, the agarose beads were washedrepeatedly and any bound virus eluted using SDS-sample buffer. Elutedproteins were separated by SDS-PAGE and HSV1 gD detected by Western Blotanalysis. This result is in agreement with previous data that M.officinalis targets HSV1 attachment to the cell.

M. officinalis has been shown to inhibit the replication of HSV1, HSV2and to a weaker extent, EHV1. To determine the spectrum of antiviralactivity associated with M. officinalis, several non-herpes familyviruses listed in Table 1 were tested for sensitivity to the extract.All assays were performed using standard plaque reduction procedures. Asshown in FIGS. 39A and 39B, M. officinalis had the strongest antiviraleffect toward HSV1 and HSV2. For this study, appropriate cell lines wereinfected with 100 pfu of the indicated virus along with treatment withincreasing concentrations of M. officinalis extract. After the formationof plaques, cells were stained with crystal violet and plaques counted.However, a second group of viruses could be inhibited by M. officinalisbut required doses approximately 10-fold higher than that required toinhibit HSV1 and HSV2. These moderately sensitive viruses includedvesicular stomatitis virus (related to rabies virus),encephalomyocarditis virus (related to polio, hepatitis A, coxsackie andecho viruses), vaccinia virus (related to variola, the causative agentof smallpox), SV40 (related to human papillomavirus) and adenovirus. Athird group of viruses were found to be insensitive to M. officinalistreatment and included mouse hepatitis virus (related to coronavirus)and reovirus (related to rotavirus).

TABLE 1 Viruses tested for sensitivity to M. officinalis botanicalextract. Related Virus pathogen Envelope Genome IC₅₀ (ml/ml) Herpesfamily + dsDNA 0.1-0.3 (HSV) 6 (EHV) VSV (Vesicular stomatitis virusRabies + ssRNA 8.2 EMCV (Encephalomyocarditis virus) Polio − ssRNA >10VACV (Vaccinia virus) Smallpox + dsDNA 3 SV40 (Simian virus 40)Papilloma − dsDNA 4.5 Reo (Reovirus) Rotavirus − dsDNA >10 Adeno(Adenovirus) Adenovirus − dsDNA 5.4Antiviral Activity of Further Botanical Extracts

Referring now to FIG. 33, anti-herpes virus activity associated withvarious Nepenthes species was examined. N. veitchii, N, spectrabilis xventricosa, N. eymae and N. Judith Finn all had antiviral activitytowards HSV1 (data not shown for N. Judith Finn). For this study, Verocells were infected with 100 pfu HSV1 followed by the addition ofincreasing concentrations of the indicated botanical extracts preparedfrom various Nepenthes species (per ml media). At 4 DPI, cells werestained with crystal violet and plaques counted N. chaniana and N. fuscahad moderate levels of antiviral activity and N. macrophylla had minimalantiviral activity.

Regarding the activity of Species II Extracts to other viruses beyondthe herpesvirus family, partial characterization of Melissa andGlycyrrhiza has been done towards SV40 virus, a member of the polyomavirus family and closely related to hitman papillomavirus (HPV). Asshown in FIG. 40, both Melissa and Glycyrrhiza extracts were able toeffectively inhibit the replication of SV40 as measured by a plaquereduction assay. In this study, Vero cells were infected with 100 pfuSV40 and treated with the indicated concentrations of M. officinalis orG. glabra extract. At 6 DPI, cells were stained with crystal violet andplaques counted. These result shown suggest that the botanical extractcomposition may not only be effective against members of the herpesvirusfamily, but will likely be a therapeutic for other viruses includinghuman papilloma virus infections. This concept based on the idea thatthe botanical extract composition contains extracts from S. purpurea, M.officinalis and G. glabra, all of which demonstrate activity againstboth herpes and polyoma viruses.

Following treatment of patients for HSV1, HSV2, and VZV with variousembodiments, it was noted that treated patients expressed a rapiddecrease in pain and discomfort associated with their lesions. Thisactivity is associated with embodiments comprising S. purpurea extract.The mechanism of action regarding this analgesic property is unknown,but adds to the therapeutic value of the S. purpurea extract byproviding pain relief along with subsequent killing of the viralpathogen and/or clearance of cancerous tissue. In addition, patientshave reported antipruritic (anti-itch) properties associated with S.purpurea following application of the extract. This has been mostnotable in patients treated for molluscum contagiosum.

Effect of S. purpurea on VACV Replication

To further determine the efficacy of S. purpurea treatment at preventingVACV replication, the ability of S. purpurea to prevent VACV inducedcytopathic effect (CPE) in vitro was examined. As used herein, “HPI”refers to hours post infection, and “MPI” refers to minutes pastinfection. Cells were infected with VACV and then treated with S.purpurea at various times post-infection. At 6 HPI, the cells wereexamined for VACV induced CPE. In untreated cells, significant VACVinduced CPE, specifically cell rounding, was observed (FIG. 2A),However, cells treated at 0, 15, and 30 MPI with S. purpurea showed noor low levels of CPE. In cells treated at 60 and 120 MPI, substantialCPE was observed, leading us to postulate that S. purpurea was likelytargeting an early component in VACV replication (i.e., viral uptake orearly viral transcription/translation). Viral uptake was monitored usinga VACV construct in which the core A5 protein was fused to cyanfluorescent protein. As shown in FIG. 2B, when the VACV infection wasperformed at 4° C., virus particles remained localized to the peripheryof the cell. When the infection was performed at 37° C., the majority ofvirus particles were observed within the cytoplasm. When a similarinfection at 37° C. was performed in the presence of S. purpurea, asimilar viral localization to the cytoplasm was observed. This suggeststhat S. purpurea treatment was not inhibiting VACV uptake into the cell.

We examined if early viral protein synthesis was inhibited following S.purpurea treatment VACV-infected cells were treated with S. purpurea atvarious times post infection and cell lysates were assayed by Westernblot using antibodies directed against total VACV proteins or the earlyVACV protein, E3L. VACV protein synthesis at 3 and 6 HPI in the cellstreated at 0, 15, and 30 MPI with S. purpurea, was greatly reduced (FIG.2C), Notably, S. purpurea reduced early VACV protein synthesis, asevidenced by the absence of the VACV-E3L protein. In contrast, S.purpurea treatment had only marginal effects on viral protein levelswhen added at 60 and 12.0 MPI. Ceils were also [35S]-methioninemetabolically labelled to compare viral protein synthesis to cellularprotein synthesis following S. purpurea treatment As predicted based onthe cellular toxicity data (FIG. 1C), S. purpurea inhibited viralprotein synthesis, whereas cellular protein synthesis remainedunaffected (FIG. 2D). Collectively, these data indicate S. purpureatreatment was effective at preventing VACV early protein accumulationand acted at a point between viral uptake and early viral proteinsynthesis.

Effect of S. purpurea Botanical Extracts on VACV Transcription In Vivoand In Vitro

Referring now to FIGS. 3A and 3B, early VACV-E3L mRNA levels werequantified in VACV-infected cells using real-time PGR to furtherunderstand the early viral stage affected following S. purpureatreatment. For FIG. 3A, HeLa cells were infected with VACV at an MO 1=10followed by the addition of 25 μl S. purpurea extract/ml media. At 4HPI, total RNA was isolated and VACV-E3L RNA levels determined byreal-time PCR. Reactions contained total RNA from mock-infected cells,ethanol/glycerol carrier-treated VACV-infected cells, or S.purpurea-treated VACV-infected cells. The term “mock-infected cells”refers to cells treated similarly as virus-infected samples, but with novirus present. C(t) values were calculated using manufacturer'ssoftware. The graph illustrates data from a representative experiment.The term “C(t)” (for “cycle threshold”) may be defined as the number ofPCR. cycles required for a fluorescent signal to cross a threshold (i.e.exceeds background level). C(t) values and fold-change from two separateexperiments are shown in Table 2.

TABLE 2 E3L gene expression in VACV-infected HeLa cells. Sample C(t)Fold-reduction VAVC (exp. 1) 13.80 VAVC (exp. 2) 10.49 VAVC + Sarracenia(exp. 1) 22.42 393 VAVC + Sarracenia (exp. 2) 19.38 474

In FIG. 3B, purified VACV virion cores were incubated in the presence ofthe indicated concentrations of S. purpurea or ethanol/glycerol carrierand [³⁵S]-UTP, Newly synthesized RNA products were spotted ontoglass-fiber filters, washed in TCA, and quantified by scintillationcounting (counts per minute). A no template reaction was performed byexcluding the addition of the virion cores.

Total RNA was isolated from cells that were mock-infected, VACV-infectedor VACV-infected followed by treatment with a single dose of S. purpureaextract. S. purpurea treatment resulted in a dramatic reduction of thelevels VACV-E3L mRNA present within the infected cells (FIG. 3A). Basedon C(t) values, the levels of early VACV mRNA were decreased by 393 to474-fold within the treated cells. No significant change in cellularactin mRNA levels was observed following S. purpurea treatment. Todetermine if VACV replication was being blocked at the level of earlyviral transcription, an in vitro transcription assay using purified VACVvirion cores and [³⁵S]-UTP was performed. The amount of VACVtranscription which occurred following treatment with increasing amountsof S. purpurea or carrier was measured by quantifying the amount[³⁵S]-UTP incorporated into the newly synthesized viral mRNA. As shown,VACV transcription decreased as the amount of S. purpurea increased,while the levels of transcription remained relatively equal in thecarrier treated cores (FIG. 38), The amount of S. purpurea required tocompletely inhibit transcription was similar to the dose that preventedVACV replication (FIG. 1B). Addition of S. purpurea to VACV cores whichhad already synthesized RNA did not reduce RNA levels suggesting thatthe extract did not have intrinsic RNase activity. Collectively, thesedata suggest that S. purpurea targets early viral transcription leadingto an inhibition in viral replication.

Spectrum of Activity

To further ascertain the efficacy of using S. purpurea, Species IIExtracts and botanical extracts to treat or prevent the symptomsassociated with a poxvirus infection, the capability of S. purpurea inpreventing the replication of more virulent members of the Orthopoxvirusgenus, namely monkeypox virus (MPXV) and variola virus (VARV) wasexamined. Referring now to FIG. 5A, HeLa cells were mock-infected orinfected with monkeypox virus at an MOI=10 followed by the addition of25 μl ethanol/glycerol carrier or 25 μl S. purpurea extract/ml media,either 0 or 15 min after infection. At 4 HPI, cell lysates were preparedand the MPXV F3L protein detected by Western blot. Referring now to FIG.4B, HeLa cells were mock-infected or infected with variola virus at anMOI=10 followed by the addition of the indicated concentrations ofethanol/glycerol carrier or S. purpurea extract to the media at 0 minafter infection. At 4 HPI, cell lysates were prepared and the VARV E3Lprotein detected by Western blot. Cells were mock-infected or infectedwith MPXV and subsequently left untreated or treated with S. purpurea orcarrier at the times indicated. Western blots for the presence of theMPXV-F3L protein, which is an ortholog of the VACV-E3L protein, wereperformed to determine if a successful MPXV infection had occurred. Whentreated with the extract at 0 or 15 MPI, S. purpurea treatment preventedthe accumulation of the MPXV-F3L protein, whereas high levels ofMPXV-F3L were detected in the both the untreated and carrier treatedcells (FIG. 5A). A similar assay was performed with VARV where cellswere mock-infected or infected with VARV and treated with S. purpurea orcarrier at the dosages indicated. Western blots for the presence of theVARV E3L protein indicated a concentration dependent inhibition in theaccumulation of the VARV-E3L protein in S. purpurea treated infections(FIG. 5B). This suggests that S. purpurea may effectively inhibit MPXVand VARV replication similarly to VACV. S. purpurea treatment alsoeffectively inhibited rabbitpox virus early protein accumulation inother experiments that have been performed. Together, these dataindicate that S. purpurea was effective at preventing the replication ofmultiple viruses within the Orthopoxvirus genus. In addition to theusefulness of S. purpurea as a therapeutic for these currently uncommoninfections, it is believed that S. purpurea may also be effectiveagainst the poxvirus, molluscum contagiosum. This is a common viraldisease with a higher incidence in children, sexually active adults, andthose who are immunodeficient.

Clinical Studies

Limited clinical studies were conducted to test the effectiveness of S.purpurea in treating infected individuals infected with molluscumcontagiosum. For these studies a 30% gel of S. purpurea was preparedusing 30 ml S. purpurea extract combined with 70 grams VERSABASE gel.This formulation was applied topically to molluscumcontagiosum-associated lesions. Application of this formulation to viralassociated lesions resulted in a slow reduction in lesion size over a3-4 week period as well as relief from itching discomfort associatedwith the infection (antipruritic activity). Three subjects have beentreated with molluscum contagiosum associated infections with similarpositive results.

(Kaposi's and molluscum): Patient diagnosed as HIV positive with AIDS.Patient has Kaposi's Sarcoma (caused by HHV 8) and molluscumcontagiosum. Patient was treated with S. purpurea (30%) in VERSABASE gel(70%) on side of face where molluscum lesions are present and to threeKS lesions. After one month, the KS lesions where were softer, flattenedout and fading. Patient also got appropriate treatment for HIV.Molluscum lesions responded well to treatment with the botanical, butpatient had them frozen off after a few weeks.

(HPV and molluscum): Patient was a 3 year old, male. Patient diagnosedwith 30+ plantar warts, common warts, seeded warts on hands, face, knee,and elbow. Diagnosed with HPV and molluscum contagiosum by apediatrician and dermatologist. Previous treatments were unsuccessful.Dermatologist applied salicylic acid to HPV and molluscum contagiosumonce a week for 3 weeks. In addition, patient applied S. purpurea (30%)in VERSABASE gel (70%) nightly with bandage. After three weeks oftreatment, warts and molluscum contagiosum lesions are non-visible. Onsubsequent follow-up examinations patient has no lesions.

(Moiluscum): Patient was a teenage male. Patient diagnosed withmolluscum contagiosum on inner thigh of both legs. Patient treated withS. purpurea (30%) in VERSABASE gel (70%) twice daily with bandage. Aftertwo weeks of treatment, molluscum contagiosum lesions were significantlysmaller. Patient also reported substantial itch relief followingapplication of the gel. Patient subsequently moved and no follow-updiagnosis is available.

In Vitro Herpes Studies

Referring now to FIG. 6, the ability of S. purpurea extract to inhibitother viruses, including herpes viruses was examined. Vero cells weremock infected or infected with HSV1 at an MOI=5. Cells were treated withS. purpurea extract at the time of infection. At 48 HPT, cells wereexamined for viral induced CPE. When cells were infected with HSV1typical cytopathic effects were observed. Following infection, if thecells were treated with S. purpurea extract, this cytopathic effectcould be virtually eliminated.

Referring now to FIG. 7, a Western blot was performed to detect thelevel of an immediate-early protein synthesized by the virus (ICP4), anearly viral protein (ICP8) and a late viral protein (gC) in order toassess at which point in the virus replication cycle S. purpurea wasacting on the HSV1. Vero cells were infected with HSV1 at an MOI=5. At 0or 2 HP1, cells were left untreated or treated with S. purpurea extract.At 24 HP1, cell lysates were prepared and viral protein levels measuredby Western Blot analysis. Antiserum used was against HSV1 ICP4, ICP8,and gC. GAPDH antiserum was used as a control. Following treatment withS. purpurea during infection, the accumulation of ICP4 was almostnon-detectable. This suggests that the S. purpurea is targeting an earlyevent in the virus replication cycle, either virus uptake into the cell,immediate-early transcription, or immediate-early protein synthesis. Iftreatment with S. purpurea is performed 2 HPI, ICP4 is detected, but theaccumulation of ICP8 is reduced and the accumulation of gC is greatlyinhibited, ICP4 synthesis was detected since the treatment with S.purpurea was begun after ICP4 had already been synthesized within thecell. This suggests that S. purpurea can also inhibit the accumulationof early and late viral proteins. Given the previous results withpoxviruses, it is believed the S. purpurea extract is likely targetingviral transcription. To confirm this inhibition of viral proteins by S.purpurea, single cycle growth studies were performed with S. purpureaextract added at various time post infection.

With reference now to FIG. 8, untreated HSV1 replicates efficiently andproduces an approximate 3.5 log increase in viral yield above inputvirus. Veto cells were infected with HSV1 at an MOI=1. At the indicatedtimes, cells were treated with S. purpurea extract for the remainingtime of the viral replication cycle. At 24 HPI, cells were harvested andviral titers determined by standard plaque assay. Input virus titerswere determined by harvesting viral infected cells at 1 hour postinfection, if the virus is treated with S. purpurea extract at 0-0.5HPI, a complete inhibition in viral replication was observed. If treatedwith S. purpurea between 1-6 HPI, viral yield is greatly reduced. Theseresults agree with the protein accumulation data of FIG. 7 where earlytreatment with S. purpurea inhibits viral protein accumulation, whereaslater treatment reduces the accumulation of late viral proteins, butsome minor synthesis was still observed.

To ascertain if this antiviral activity was limited to HSV1, activityagainst Varicella-Zoster virus (VZV) was tested. Referring now to FIG.9, BSC1 cells were infected with VZV at a low MOI. As indicated cellswere left untreated or treated with S. purpurea extract. Viral plaqueformation was monitored and shown at 7 and 9 DPI. Again, infection ofcells in culture with VZV leads to a distinct cytopathic effect.Treatment of infected cells with S. purpurea extract immediately afterinfection was able to completely abolish this cytopathic effect (FIG.9). Notably, these assays were performed with a single dose of S.purpurea extract (on day 1) and cytopathic effects measured on days 7and 9.

Referring now to FIG. 10, individual plaques formed by VZV in themonolayer were observed by staining the cell monolayer to quantitatethis effect. In this experiment, BSC1 cells were infected with VZV at alow MOI. As indicated, cells were left untreated or treated with 10μl/ml or 20 μl/ml S. purpurea extract. At 9 and 12 DPI, cell monolayerswere stained with crystal violet. The presence or absence of plaqueformation is shown at 9 DPI. No plaque formation was detected for theuninfected control at either 9 or 12 DPI. At 9 DPI, the untreated virusinfected cell monolayer had 288 plaques, and at 12 DPI, over 300 largeplaques were present. As shown, a dramatic inhibition in plaqueformation was observed following treatment with the S. purpurea extract.At 9 DPI, neither the 10 μl/ml or 20 μl/ml S. purpurea extract treatedexperiments had detectable plaques. At 12 DPI, about 40 small plaqueswere counted for the 10 μl/ml S. purpurea extract treatment, while the20 μl/ml S. purpurea extract treatment had no detectable plaques. Thisinhibition in the replication of VZV by S. purpurea was confirmed byWestern blot to detect the gI protein of the virus.

As shown in FIG. 11, this protein could not be detected in cellsinfected by VZV which were treated with S. purpurea extract. BSC 1 cellswere infected with VZV at an MOI=0.01. Cells were left untreated ortreated with S. purpurea extract. At 12 DPI, cell lysates were preparedand the level of VZV gl expression determined by Western Blot analysis.Together these results suggest that the S. purpurea extract caneffectively inhibit the replication of HSV1 and VZV, and it is likely toinhibit other members of the herpesvirus family as well.

S. purpurea extract was also tested against the related herpes simplex-2(HSV-2) and equine herpes virus-1 (EHV-1). Referring now to FIG. 12, S.purpurea extracts demonstrated similar antiviral activity against bothHSV-2 and EHV-1 similar to that observed toward HSV-1. Vero cells wereinfected with HSV 1, HSV2 or EHV1 with 100 pfu/well. At the time ofinfection, cells were treated with various concentrations of S.purpurea, as indicated in the chart. At four DPI, cell monolayers werestained with crystal violet and plaque numbers counted. Plaque formationis shown as a percentage of a cell monolayer exhibiting the presence ofplaques.

Clinical Herpes Studies

Clinical studies were conducted to test the effectiveness of S. purpureain treating individuals with herpes virus (HSV1). For these studies a20% gel of S. purpurea (“20% Gel”) was prepared using 20 ml S. purpureaextract combined with 80 grams VERSABASE gel. Subjects diagnosed withHSV1 lesions on their lips were treated 4 times daily with 20% Gel.Lesion size and progression was monitored and photographed daily.

FIGS. 13A-13D illustrate the effects of the 20% Gel treatment for atypical subject. Viral lesions are indicated by arrows. As illustratedin FIGS. 13A-13D, over the course of the treatment period, the size andprominence of the viral lesions decreased. At the outset of treatment(Day 0, illustrated in FIG. 13 A) the lesion on the left was large andprominent. As shown in FIG. 13B (Day 1), after the first day oftreatment, the lesion on the left and decreased substantially in size.On Day 2 (FIG. 13C), both lesions showed further healing, and by Day 3of treatment (Figured 13D), the lesions were substantially healed.

Approximately 500 patients with similar HSV1 oral lesions were treatedwith similar results (over a 95% success rate compared to untreated HSV1lesions). For topical therapy, 20% Gel was applied topically toviral-associated lesions. Application of 20% Gel to HSV 1 associatedoral lesions (“cold-sore”) every 4 hours over the course of infectionsled to rapid recovery of lesions FIG. Other herpes virus-associatedinfections were treated in patients, including infections withgenital-associated HSV2 lesions and VZV-associated lesions. For thepatients studied, successful effects following 20% Gel treatment wereobserved, suggesting an inhibition of virus replication within thetissue. For VZV patients, the formulation was applied to using anabsorbent pad or impregnated bandage which could then be applied to thepatient to cover afflicted areas.

In addition, some herpes virus family members are known to be associatedwith the development of cancers. Specifically Epstein Barr virus (EBV)and human herpes 8 (HHV8) are known to cause jaw/throat/neck cancer andKaposi's sarcoma, respectively. A select number of subjects with suchviral induced cancers were treated with success. Results of a few ofthese cases are shown herein.

(EBV/HHV6): Patient is a 57 year old male diagnosed with carcinoma ofthe pharynx. There is a right-sided posterior pharyngeal mass. Patientnoticed a “bump on tongue” and was diagnosed via CT. Patient had aright-sided submandibular node palpated externally and on left pharynxon oral cavity exam in the peritonsilar region. The mass was accessibleonly by cotton swab. Patient reported mild discomfort in swallowing, butnot enough to interfere with activities of daily living. No painreported. Patient was diagnosed as HHV6+(IgG) and EBV+ (IgO). Patientwas treated with S. purpurea liquid (50%) with an equal part transdermaldriver gel (50%) applied directly to the peritonsilar/pharyngeal lesion.This regimen was incorporated as a part of an integrative treatmentprotocol. Upon administering directly to the tumor mass, patientreported that “the tumor just vanished under the swab as I was applyingit.” Within two days, the pharyngeal tumors were significantly smaller,and were visibly collapsing under the superficial mucosa, first in astar-like pattern. The mass became non-evident with remnants of scartissue. At the time of discharge, patient reported that he had notrouble swallowing and was only feeling what felt to be scar tissue. Nopain or bleeding rioted at the time of discharge. During treatment,PET/CT revealed interval decrease in size of tumor. For follow-up, twomonths after discharge patient received a clean PET/CT.

(EBV): Patient was an 8 year old male with aphthous ulcers on tongue andoral mucosa. Physical diagnosis indicates lesions likely due to EBVinfection with the development of hairy leukoplakia. Symptoms appear tobe stress related and the pain intensity to the level that the patientcan't attend school nor eat without discomfort. Patient was treated withS. purpurea liquid (50%) as a gargle four times daily. With treatmentpatient's symptoms gradually improved. Improved symptoms were riotedwithin a few days with complete recovery after 2 weeks.

(Kaposi's and molluscum): Patient diagnosed as HIV positive with AIDS.Patient has Kaposi's Sarcoma (caused by HHV 8) and molluscumcontagiosum. Patient was treated with S. purpurea (30%) in VERSABASE gel(70%) on side of face where molluscum lesions are present and to threeKS lesions. After one month, the KS lesions where were softer, flattenedout and fading. Patient also received appropriate treatment for HIV.Molluscum lesions responded well to treatment with the botanical.

In Vitro Polyomavirus Studies

Referring now to FIG. 14, Vero cells were infected with SV40 virus at anMOI=1. Ceils were treated with S. purpurea extract as indicated in thecaptions above the photographs. At four DPI, viral induced CPE wasvisualized by microscopy and photographed. After assessing the abilityof S. purpurea extracts to inhibit poxvirus replication, the inhibitoryactivity against other, non-related viruses, including polyoma andpapillomaviruses was also examined. Since papillomaviruses cannot beused for in vitro studies, the in vitro viral infection work was doneusing a related virus, simian virus 40 (SV40), which belongs to thefamily of polyoma viruses. When cells were infected with SV40 typicalcytopathic effects were observed. In cells which were infected with SV40followed by-treatment with the S. purpurea extract, the cytopathiceffect was inhibited (FIG. 14).

To confirm this effect as a block in virus replication. Western blot wasperformed to detect the synthesis of viral proteins, as shown now inFIG. 15. Vero cells were infected with SV40 at an MOI=1. Cells weretreated with varying concentrations of S. purpurea extract as indicated.At 24 and 72 HPI, cell lysates were prepared and levels of viral proteinaccumulation measured by Western Blot analysis. Viral antisera includedSV40 LargeT and VP1. Antiserum to GAPDH was used as a control.

The accumulation of SV40 LgT and VP1 proteins was inhibited by treatmentwith S. purpurea, Notably, since the LgT protein is an early proteinmade by the virus, this data again suggests that S. purpurea istargeting an early event in the virus replication cycle, either viraluptake, viral transcription, or viral protein synthesis. Given theresults of experiments with vaccinia virus, the likely mechanism ofaction is proposed to be an inhibition of viral transcription. Theseresults suggest that S. purpurea extract can inhibit the replication ofpolyoma and likely papillomaviruses. Given this, S. purpurea may be aneffective therapeutic for infections or cancers caused by these familiesof viruses.

Referring now to FIGS. 16A-16E, since S. purpurea extracts inhibited theaccumulation of early proteins during an SV40 virus infection, theeffect of S. purpurea extracts on cervical cancer cells was determined.SiHa (cervical cancer cell line) cells were plated into 60 mm dishes ata 1:10 confluency (day 1). Duplicate plates were treated with theconcentrations of S. purpurea extract indicated in the figures. Six dayspost treatment, cells were evaluated by microscopy and photographed.SiHa cells are a cervical cancer cell line which was isolated from apatient infected with Human papilloma virus (HPV 16). These cellsconstitutively synthesize the viral proteins E6 and E7 which areresponsible for leading to cell division and cancer. When SiHa cellswere treated with increasing doses of S. purpurea extract, an inhibitionin cell division (senescence) was observed at lower doses, and celldeath (likely due to apoptosis) was observed at higher doses.

Referring now to FIGS. 17A-17D, higher magnification views of cellstreated with various doses of S. purpurea extract and demonstratingcytoplasmic vacuole formation and ‘blebbing’ typically indicative ofapoptosis are shown.

With reference now to FIGS. 18A-19B, cells treated with S. purpureademonstrated reduced levels of HPV E6 and HPV E7 protein synthesis.FIGS. 18A-19B illustrate western blot results for HPV E6 and E7 proteinlevels in SiHa cells treated with the indicated concentrations of S.purpurea extract at 24 and 48 hours post treatment. Cell lysates wereprepared from the treated SiHa cells at the two time points indicated.Antiserum to GAPDH was used as a control.

HPV E6 protein induces cell division by stimulating ubiquitination ofthe cellular p53 protein, thereby promoting p53 degradation. Since HPVE6 levels decreased following S. purpurea treatment, the levels of p53protein were assessed.

Referring now to FIGS. 20A and 20B, treatment of SiHa cells with S.purpurea led to increased levels of p53 protein at 24 and 48 hours posttreatment SiHa cells were treated with the concentrations of S. purpureaextract indicated. At 24 and 48 hours post treatment, cell lysates wereprepared. Cellular p53 protein levels were evaluated by western blotanalysis. The results of the western blot were quantified by ImageQuantanalysis (FIG. 20B) to measure the relative pixel intensity of thedetected protein. These results support that S. purpurea may act byreducing HPV E6 (and E7) levels, leading to a recovery in cellular p53levels and a subsequent inhibition in cell division and induction ofapoptosis.

With reference now to FIG. 21, in order to quantitate an effect of S.purpurea on cellular senescence, cell division was monitored for SiHacell treated with a low dose of S. purpurea extract. SiHa cells wereplated into 60 mm dishes at a 1:10 confluency. Cells were left untreatedor treated with 5 μl/ml S. purpurea extract Medium containing 5 μl/ml S.purpurea extract was replaced daily for the S. purpurea treated cells.Every 24 hours, cells were harvested and counted using a hemocytometer.Cell numbers were monitored for a period of 7 days. As shown in FIG. 21,S. purpurea treatment significantly inhibited cell division compared tovehicle treated cells. These results together suggest that S. purpureaextract inhibits the synthesis of HPV E6 and E7 proteins which likelyleads to the induction of cellular senescence and apoptosis. Theseresults support the possible therapeutic value of S. purpurea extractsfor treatment of HPV-induced cervical cancer.

Clinical Papillomavirus Studies

Since papillomaviruses cannot be used for in vitro studies, butrepresent a significant human pathogen, clinical studies were performedusing a few patients infected with human papillomaviruses. To date,limited number of patients infected with genital HPV (HPV 16/18) andHPV-associated plantar warts have been treated topically with the 20% S.purpurea Gel. Patients treated demonstrated positive results followingtreatment with 20% S. purpurea Gel. For genital HPV infections, 20% S.purpurea Gel was applied topically as the aforementioned gel as well asin suppository form (containing Supposiblend® (Gallipot®), from Fagron,St. Paul, Minn., Silica Gel Micronized NF® (Letco Medical, Decatur,Ala.) and the S. purpurea extract). For plantar warts, success rateswere improved if the outer epidermal layer was initially removed withfreezing followed by application of the S. purpurea gel. These resultstogether support an extract of S. purpurea as being a potentialtreatment and therapy for the infections related to papilloma andlikely, polyomavirus infections.

HPV is accepted as being the causative agent for nearly all cases ofcervical cancer. The development of cervical dysplasia is thedevelopment of precancerous changes to the cells lining the cervix. Inaddition, HPV is associated with other epithelial cancers of the anusand oral cavity. Finally, HPV has been suggested to be involved in thedevelopment of actinic keratosis and squamous cell carcinoma of theepidermis.

(HPV possible): Patient is a 62 year old male diagnosed with squamouscell carcinoma of the tongue. Patient reports having had intense jawpain and was on multiple antibiotics. Shortly thereafter, patient founda lump in the throat. Biopsy was performed confirming squamous cellcarcinoma. At that time, the physician recommended surgery but the lumpwas considered inoperable by the surgeon. At the time of initial intake,patient was losing weight due to not being able to eat any solid foods,and not being able to swallow due to the pain coming from the tumor. Onphysical examination there was a right-sided, erythematous lesion on thebase of the tongue. Pain was rated at an 8/10. Patient was treated witha therapeutic composition comprising S. purpurea liquid (50%) with anequal part transdermal driver gel (50%). Driver was equal partspleuronic gel and VERSABASE gel. This formulation applied directly bypatient to the tumor four times daily was incorporated as a part of anintegrative treatment protocol. After four days of treatment, palpabledecrease in tumor diameter. After one week, patient reported no pain.After 10 days, tumor was reported to be softening up to the touch. After1 month, tumor significantly reduced in size. After 3 months, tumor wasno longer detectable and had become scar tissue.

(HPV possible): Patient is a 71 year old male diagnosed with squamouscell carcinoma of the larynx. Squamous cell carcinoma of larynxdiagnosed in 2010. Patient treated with radiation therapy at that time.Patient was in remission until a year later when it returned. Patientstates that he progressively lost his voice throughout this process andis now seeking integrative cancer care. Upon physical exam, larynx wasvisibly and palpably enlarged at time of initial examination. Patientwas treated with a therapeutic composition comprising S. purpurea liquid(50%) with an equal part transdermal driver gel (50%). The driver wasequal parts pleuronic gel and VERSABASE gel This formulation was applieddirectly by the patient to the outer surface of the neck in the regionof the larynx as well as gargled four times daily. This regimen wasincorporated as a part of an integrative treatment protocol. Within afew days, patient reported increased ability to talk with a more audibleand discernible voice. Within 1 week, he was able to speak for most ofthe day without having to rest his voice as often, and reported that hisvoice sounded clearer and more people were able to understand himwithout him having to force his vocal cords. Within 3-4 weeks at thetime of discharge, patient was reporting increased energy, speakingaudibly and mild hoarseness. Indications of squamous cell carcinoma werenon-detectable.

(HPV likely): Patient is a 61 year old female diagnosed with squamouscell carcinoma of the anus. Upon initial examination, patient reportedthat she noticed some growth on the outside of her anus. Uponexamination, an external anal mass measuring 14.5×14.5 cm was observed.The mass was freely mobile and soft on palpation. An anal fissure wasappreciated at the 12:00 position about 2-3 mm deep and 3 mm wide.Patient reported at the time of exam that the mass was previously veryhard and immobile. Patient was treated with a therapeutic compositioncomprising S. purpurea liquid (50%) with an equal part transdermaldriver gel (50%). The driver was equal parts pleuronic gel and VERSABASEgel. This formulation was applied directly by patient to the tumor via arectal implant 2-times daily. A 60 cc TERUMO® syringe (Terumo MedicalCorporation, Somerset, N.J.) with a round Robinson catheter was utilizedfor the application. This regimen was incorporated as a part of anintegrative treatment protocol. Within one week of treatment, patientreported decreased size of the mass that was palpable by self, and alsoexperienced decreased anal bleeding and absence of pain. Patient alsoreported that mucous-like stringy filaments were being released with bowel movements and during colon hydrotherapy. A sample was sent forcytology and came back as mucous only. By the time patient wasdischarged for part time status due to personal reasons, she reported nopain, ease of insertion of speculum during colonic irrigation, regular,w ell formed bow el movements and no pain or bleeding.

(HPV dysplasia): Patient is 34 year old white, female. She testedpositive for HPV and cervical dysplasia three years prior. Colposcopywas done and patient was put on a ‘watch and wait’ with follow-upsscheduled. Upon re-exam, patient was positive for dysplasia (CIN1).Patient was treated with a therapeutic composition comprising S.purpurea liquid (50%) with an equal part transdermal driver gel (50%)applied once a week to the face of the cervix with the assistance of herdoctor as well as self-application digitally to her vaginal wall twicedaily for 4 wks. At this time, she continued with application oftreatment to the face of the cervix by her doctor once a week for twomore weeks and self-administration of a suppository containing thetreatment daily for two additional weeks. After a period of one month,patient was re-examined. Examination results were negative for HPV andASCUS.

(HPV warts and molluscum): Patient was a 3 year old, male diagnosed with30+ plantar warts, common warts, seeded warts on hands, face, knee, andelbow. Patient was diagnosed with HPV and Molluscum Contagiosum bypediatrician and dermatologist. Previous treatments were unsuccessful.Dermatologist applied salicylic acid to HPV and Molluscum Contagiosumonce a week for 3 weeks, in addition, patient applied a therapeuticcomposition comprising S. purpurea (30%) in VERSABASE gel (70%) nightlywith bandage. After three weeks of treatment, warts and molluscumcontagiosum lesions are non-visible. On subsequent follow-upexaminations patient has no lesions.

(HPV warts): Patient was a 28 year old female diagnosed with a large(1.5 cm) Verruca plantaris (plantar wart) by a dermatologist. Surgicalremoval was suggested but adjunctive usage of a therapeutic compositioncomprising S. purpurea (30%) in VERSABASE gel (70%) was requested by thepatient. Her dermatologist performed one cryogenic treatment and patientapplied the therapeutic composition twice daily with a bandage. Patientreported pain relief associated with application of the therapeuticcomposition. After 2 months, wart completely resolved and has notreturned.

(HPV genital warts): Patient diagnosed HIV disease and significant HPVwarts on vaginal vulvae, especially on left side. Pap smear history wasunknown. Patient was treated with a therapeutic composition comprisingS. purpurea (30%) in VERSABASE gel (70%). Warts were successfullytreated with botanical gel.

(HPV): Patient diagnosed with HIV. Patient had a single HPV wart on theanal verge. Patient was treated with a therapeutic compositioncomprising S. purpurea (30%) in VERSABASE gel (70%). Treatmentsuccessfully eradicated the wart. Post-treatment, the wart started toreappear. Treatment was restarted for the next month. Wart waseliminated and has not returned

Actinic keratotis (HPV possible): Patient was a 42 year old femalediagnosed with actinic keratosis lesion on left side of face. Patientscheduled for surgical removal of the lesion 10 days after diagnosis.While awaiting surgery, patient applied a therapeutic compositioncomprising S. purpurea (30%) in VERSABASE gel (70%) four times daily tothe lesion. After seven days of treatment, lesion was non-visible.Patient subsequently cancelled surgery. initial studies have beenconducted to characterize potential anti-carcinogenic activityassociated with extracts of S. purpurea. In vitro data supportsanticancer activity associated with S. purpurea with the SiHa cervicalcancer cells. In patients with diagnosed cancerous or pre-cancerouslesions, treatment with the S. purpurea gel has demonstrated positiveresults. As described above, S. purpurea gel has been shown effective inthe treatment of HPV associated cervical dysplasia and warts. Inaddition, several patients diagnosed with squamous cell carcinomafollowing treatment with the S. purpurea gel (applied for 1 week to 2months depending on severity) had typical positive results includedclearing of the lesions and biopsy negative diagnoses. In addition,several patients diagnosed with a pre-cancerous actinic keratosis lesionfollowing treatment with S. purpurea gel resulted in complete clearanceof the lesion typically within 1-2 weeks of application. We have alsoused S. purpurea gel in the treatment of HPV and Epstein-Barr associatedoral-pharyngeal carcinomas. External topical or topical within the oralcavity has reduced the size of solid mass tumors present in severalpatients. These results suggest that an extract from S. purpurea haspotential anti-carcinogenic activity.

Possible Active Constituents)

Referring now to FIGS. 23A-23C, tests with purified betulin supportsimilar antiviral activity compared to S. purpurea extracts. Hela cellswere infected with 100 pfu VACV in the presence of increasingconcentrations of purified betulin, lupeol or betulinic acid. Thestructures of betulin, lupeol and betulinic acid are shown in FIGS. 23A,23B, and 23C, respectively. At 48 HPI, cells were stained with crystalviolet and the number of plaques formed counted. Duplicate plates wereleft uninfected but treated with the same concentrations of betulin,lupeol, or betulinic acid. Cell viability was evaluated 24 hours posttreatment by trypan blue exclusion. Results of the antiviral activityand cell viability assays are illustrated in FIGS. 24A-24C. Relatedcompounds, lupeol and betulinic acid did not show suitable antiviralactivity. These results may suggest betulin as at least one antiviralconstituent present in Sarracenia spp.

Biological Assays

Referring now to FIGS. 25A-62B, further work was done to develop ascreening methodology to identify additional botanical-based extractswith efficacy against viruses, in particular herpes viruses. FIGS.25A-25C illustrate the results of an experiment in which Vero cells wereinfected with 300 pfu HSV1, followed by treatment with increasingconcentrations of a botanical extract to be evaluated. At four DPI,cells were stained with crystal violet to visualize plaque/CPE. Controlplates were left uninfected (FIG. 25A), while treatment plates weretreated with increasing dose amounts of eithera non-effective botanicalextract or an effective botanical extract. 24 hours later, cellviability determined by trypan blue exclusion (data not shown). Asshown, non-effective botanical extracts (FIG. 25B) do not inhibitplaque/CPE formation by the virus. Effective botanicals (FIG. 25C)inhibit viral replication in a dose dependent manner which can bequantified and scored. Comparable effects on viability can also bescored (not shown). In FIGS. 26A and 26B, using the method describedabove with respect to FIG. 25, additional botanical extracts showingmoderate antiviral activity (FIG. 26A) and high antiviral activity (FIG.26B) were further evaluated quantitatively and qualitatively bymeasuring the effect of the botanical extract on plaque formation,plaque numbers and cell toxicity (not shown). Botanical extracts can bescored and ranked accordingly as non-effective, moderately effective,highly effective or various values in between.

These botanical screens identified five (5) additional botanicalextracts with antiviral activity against HSV1. Botanical extracts fromMelissa officinalis, Lavandula officinalis, Glycyrrhiza glabra,Eleutherococcus senticosus and Hypericum perforatum were blended with S.purpurea,

The resulting botanical extract composition created a formulationcapable of inhibiting herpes virus replication and demonstratingeffective therapeutic value in the treatment of alpha herpes virusinfections in patients, including HSV1 associated herpes labialis (‘coldsores’), HSV2 genital infections, and varicella-zoster virus associatedshingles. In addition, this composition demonstrated analgesic(anti-pain) and antipruritic (anti-itch) properties toward the relief ofdiscomfort associated with these conditions.

Cell monolayers of Vero cells were seeded the day prior to infection tobe at 90% confluency the following day. HSV1 (KOS strain) was seriallydiluted to a final concentration of 200 plaque forming units/100 μl.Botanical extracts to be tested for antiviral activity were filteredthrough a 0.2 μm syringe filter (to remove microbial contaminants),evaporated under vacuum to remove excess ethanol and the centrifuged toremove any precipitates. Viral solutions were pre-treated withincreasing concentrations of the individual extracts for 20 min and thenthe Vero cell monolayers were infected with these treated samples for 30min. Growth media was then added to the cell dishes and additionalbotanical extract added to the media at the appropriate concentration(for which the viral solutions were treated). The cells were incubatedat 37° C. for 48 hours and then stained with crystal violet to visualizeplaque formation. Plaques were counted and viral inhibitory units(VIU)/ml calculated (1 VIU equals the concentration of the extractrequired to inhibit viral replication by 50%). In parallel, Vero cellmonolayers were left uninfected, but treated with increasingconcentrations of the botanical extracts. Twenty-four hours later, cellviability was determined by standard trypan blue exclusion procedures.From this, the cell cytotoxic concentration could be determined (CC50equals the concentration of the extract required to kill 50% of the cellpopulation). Based on the VTU and CC50 values, the specificity index ofeach botanical extract could be determined. Botanical extracts could beidentified which had no, moderate or high antiviral activity (FIGS.27-29).

From the botanical screening procedure, six extracts with anti-HSV1activity were identified, including Sarracenia purpurea, Melissaofficinalis, Lavandula officinalis, Glycyrrhiza glabra, Eleutherococcussenticosus and Hypericum perforatum. As shown in FIGS. 27-29, thesebotanicals demonstrated antiviral activity towards HSV1 and comparablylow cell cytotoxicity. In FIG. 2.7, the specificity of S. purpureaeffect on viral replication in comparison to cell toxicity isillustrated. For viral replication, viral translation levels (closedsquares) were measured. HeLa cells were infected with VACV at an MOI=10followed by the addition of the indicated concentrations of S. purpureaextract/ml media. At 6 HPI, cell lysates were prepared, the VACV E3Lprotein detected by Western blot, and quantified. For cell viability,HeLa cells were treated with the indicated concentrations of S. purpureaextract (closed squares) or ethanol/glycerol carrier (closed triangles)for 6 hours and the number of viable cells determined by a trypan blueexclusion assay. In FIG. 28, the specificity of M. officinalis extracton viral replication in comparison to cell toxicity was evaluated. Forviral replication, Vero cells were infected with 100 pfu HSV1 followedby the addition of the indicated concentrations of M. officinalisextract/ml media. At 4 DPI, cells were stained with crystal violet andplaques counted. For cell viability, Vero cells were treated with theindicated concentrations of M. officinalis extract or ethanol/glycerolcarrier for 24 hours and the number of viable cells determined by atrypan blue exclusion assay. In FIGS. 29A-29D, the method described inFIG. 2.7 was used to evaluate the specificity of G. glabra (FIG. 29A),L. officinalis (FIG. 29B), H. performatum (FIG. 29C) and E. senticosus(FIG. 29D) extracts relative to antiviral activity and cell toxicity.

Since human infection with alpha viruses (HSV1, HSV2 andvaricella-zoster virus) typically present with lesions on the epidermis,therapeutic effects of the botanical extract composition may be improvedby suspension of the aqueous extract in a gel to provide topicalapplication and transdermal ‘driving’ capabilities, as described herein.For this application, a gel suspension was prepared using a 50% aqueousbotanical extract composition combined with 50% VERSABASE gel.

An embodiment of a therapeutic composition used for topical treatment ofherpes virus infections and comprising a botanical extract compositionis shown in Table 3.

TABLE 3 Therapeutic composition of botanical extracts used for topicaltreatment of herpes virus infections. Percent of Component compositionmls/100 mls VIU/ml Sarracenia purpurea 25 25 25 Melissa officinalis 6 640 Lavendula officinalis 10 10 10 Glycyrrhiza glabra 2.5 2.5 20Hypericum performatum 2.5 2.5 20 Eleutherococcus senticosus 4 4 15Versabase 50 50As illustrated in Table 3, in various embodiments, a botanical extractcomposition may comprise 25% Sarracenia purpurea (25 viral inhibitoryunits (VIU)/ml), 6% Melissa officinalis (40 VIU/ml), 10% Lavandulaofficinalis (10 VIU/ml), 2.5% Glycyrrhiza glabra (20 VIU/ml), 2.5%Eleutherococcus senticosus (20 VIU/ml), and 4% Hypericum perforatum (15VIU/ml). The botanical extract composition thus comprises all Species IIExtracts. In various embodiments, the proportions of the extracts andbase gel may be modified to the adjust biological activity or thesuitability of the composition for various applications.Individual Extract Activity Toward Alpha Herpes Viruses

The antiviral activity of each of the Species II extracts was tested forefficacy against HSV1, HSV2 and EHV1. As shown in FIGS. 30A-30F, all sixextracts had antiviral activity against HSV1 and HSV2. Vero cells wereinfected with 100 pfu HSV1, HSV2 or EHV1 followed by the addition ofincreasing concentrations of the indicated botanical extracts (per mimedia). At 4 DPI, cells were stained with crystal violet and plaquescounted.

Most botanical extracts actually demonstrated slightly stronger activityagainst HSV2. For EHV1, Sarracenia purpurea (FIG. 30A) had strongantiviral activity; Melissa officinalis (FIG. 30B), Lavandulaofficinalis (FIG. 30C), Glycyrrhiza glabra (FIG. 30D), and Hypericumperforatum (FIG. 30E) all had moderate antiviral activity; andEleutherococcus senticosus (FIG. 30F) had no detectable activity againstEHV1. These results suggest that, individually, the botanical extractshave effective antiviral activity against the human alpha herpesviruses, including HSV1 and HSV2.

Synergistic Antiviral Activity of Combined Botanical Extracts

Since the Species II Extracts present in the botanical extract blendhave antiviral activity in the blend as well as individually, theSpecies II Extracts were tested in various pairwise combinations toassess potential synergistic activity. As shown in FIGS. 31A and 31Bwhen assessing reductions in plaque numbers by different botanicalextract combinations, several combinations of botanical extractsresulted in positive synergistic antiviral effects, as shown by the (+)in the figure. For the study shown in FIGS. 31A and 31B, Vero cells wereinfected with 1000 pfu HSV1 followed by the addition of the indicatedbotanical extracts individually or various paired combinations. Dosesused were at 50-75% of the 100% viral inhibitory concentration. At 4DPI, cells were stained with crystal violet and plaques counted. FIG.31A illustrates the data of FIG. 31B in bar graph form.

A synergistic effect exists whenever the action of a combination isgreater than the sum of the activity of the individual compounds. Invarious embodiments, a synergistic effect exists when a combination ofbotanical extracts shows a greater antiviral effect than one wouldpredict from the sum of the individual effects of the botanical extractsused in the combination. The action E to be expected for a given activeingredient combination, e.g. the combination of two botanicals, obeysthe COLBY formula and can be calculated as follows (COLBY, S. R.,“Calculating synergistic and antagonistic responses of herbicidecombination”. Weeds, Vol. 15, pages 20-22; 1967):

In the COLBY formula, X=action by active ingredient I; Y=action byactive ingredient II, and E=the expected action of active ingredientI+II, and whereinE=X+Y(XY/100)O is the observed action, and O/E=SF, the Synergy Factor.

If the action (O) actually observed is greater than the expected action(E), then the action of the combination is superadditive, i.e., there isa synergistic effect quantified as the Synergy Factor, SF. In this case,positive synergistic effects were observed when plaque reductions weregreater then the predicted reduction values based on treatments with theindividual Species II Extracts alone. As an example, if a single SpeciesII Extract were used at a dose which inhibited viral replication by 90%,10% of the uninhibited virus would still replicate and spread to newcells. If two Species II Extracts were combined which have additiveeffects and both used at a dose which inhibited 90% of viralreplication, 99% of the virus would be inhibited. If however thesebotanicals have positive synergistic activity, inhibition is evenhigher. Similarly, when assessing plaque size, several botanical extractcombinations provided positive synergistic effects.

As shown in FIGS. 31A and 31B, a synergistic effect was seen inparticular combinations when measuring virus inhibition by plaquenumber. When measuring inhibition by plaque number (FIG. 31 A), 7 of the15 pair wise botanical combinations of Species II Extracts producedadditive antiviral effects. However, 7 of the 15 pairs producedsynergistic effects. The pairs of botanicals showing synergistic effectincluded: Melissa+Sarracena; Melissa+Glycyrrhiza;Sarracenia+Eleutherococcus; Sarracenia+Glycyrrhiza;Sarracenia+Lavandula; Eleutherococcus+Glycyrriza; Glycyrhizza+Lavandula.As can be further seen in FIGS. 31A and 31B, when measuring virusinhibition by plaque size, 4 of the 15 Species II Extract combinationsproduced additive effects. However, 8 of the 15 pairs of Species IIExtracts showed synergistic effect. In the plaque size experiments (FIG.31B), the pairs showing synergistic effect included: Melissa+Sarracenia;Melissa+Eleutherococcus; Sarracenia+Eleutherococcus;Sarracenia+Glycyrrhiza; Sarracenia+Hypericum; Sarracenia+Lavandula;Eleutherococcus+Hypericum; and Eleutherococcus+Lavandula.

As shown in FIGS. 31A and 31B, a synergistic effect was seen inparticular combinations when measuring virus inhibition by plaquenumber. When measuring inhibition by plaque number, 10 of the 15 pairwise botanical combinations of Species II Extracts produced additiveantiviral effects. However, 4 of the 15 pairs produced synergisticeffects. The pairs of botanicals showing synergistic effect included:Melissa+Glycyrrhiza; Sarracenia+Eleutherococcus; Sarracenia+Glycyrrhiza;and Sarracenia+Lavandula. Also shown in FIGS. 31A and 31B, whenmeasuring virus inhibition by plaque size, 5 of the 15 Species IIExtract combinations produced additive effects. However, 7 of the 15pairs of Species II Extracts showed synergistic effect. In the plaquesize experiments, the pairs showing synergistic effect included:Melissa+Eleutherococcus; Sarracenia+Eleutherococcus;Sarracenia+Glycyrrhiza; Sarracenia+Hypericum; Sarracenia+Lavandula;Eleutherococcus+Hypericum; and Eleutherococcus+Lavandula.

In view of these results, other pairs of botanical extracts, and morecomplicated blends such as those incorporating three or more botanicalextracts, may likewise show synergistic antiviral effects.

Additionally, the combination of Sarracenia and Drosera extracts wasfound to dramatically inhibit the replication of members of the poxvirusfamily. This was demonstrated in various experimental methodologiesincluding a reduction in the viral growth level, an inhibition in theability of the virus to form plaques, a block in viral proteinsynthesis, and an inhibition in viral induced cell killing. Reduction invirus yield was approximately 90% with a single treatment,

Clinical Herpes Studies

Limited clinical studies were conducted to test the effectiveness of abotanical extract composition as disclosed herein in treating alphaherpes virus infected individuals. Table 4 lists the number of subjectswho were diagnosed with herpes family virus infections and subsequentlytreated with the botanical extract composition described in Table 3.Approximate success rates of therapeutic treatment are indicated.

TABLE 4 Conditions and number of patients treated Condition Number ofpatients Approx. success rate HSV1 cold sores >500  95% HSV2 genitallesions 25 100% VZV (Shingles) 18  95% Epstein Barr 2 100%

For these studies, a gel-based therapeutic composition of a botanicalextract composition was prepared as described herein. Since theseinfections presented as epidermal lesions, therapy was done topically.The gel formulation was applied directly and topically toviral-associated lesions. For HSV1, treatment was given to subjectsdiagnosed with herpes labialis (oral ‘cold sores’). Subjects applied theMelivir gel topically every 4 hours over the course of the infection.Subjects were required to clean the lesion area prior to application ofthe gel and were NOT allowed to apply cosmetics or other topicalgels/creams to the area. For subjects treated in the macule/erythema,vesicle or papule stage, a decrease in inflammation was typicallyobserved within 12-18 hours and scabbing within 24-48 hours. Completehealing was observed in most subjects within 3-5 days followingtreatment. For subjects treated in the prodrome stage (prior to eruptionof a lesion), typical results recorded an inhibition of progression ofsymptoms and no eruption of a visible lesion. To date, approximately 500subjects with similar HSV1 oral lesions with similar results (over a 95%success rate compared to untreated HSV1 lesions). Notably, for mostsubjects that had previously reported common HSV1 outbreaks (every 1-3months), repeated treatments with the gel formulation during subsequentoutbreaks were required less frequently, with a greater time periodbetween outbreaks.

We have also treated other herpes virus-associated infections withsubjects including genital-associated HSV2 lesions (25 subjects). Forgenital treatment, the gel formulation was applied topically every 4hours along with a suppository therapeutic composition comprising thesame botanical extract composition. Significant healing of the infectedtissue was observed within 24-48 hours with complete healing typicallyin 4-5 days.

For varicella-zoster (VZV) subjects (18 subjects) presenting withshingles symptoms, the gel formulation was applied using an absorbentpad or impregnated bandage which could then be applied to the patient tocover afflicted areas. This patch allowed the gel to remain localized tothe afflicted area without being removed by clothing or rubbing. Similarto HSV1 and HSV2, healing of the lesions was observed within a few days(typically 4-7 days) following application of the gel. Typical resultsfor a VZV patient treated with the gel formulation are shown in FIGS.41A-41C. In this study, the subject was diagnosed with a VZV lesion onthe left side of nose. The subject had severe lesion with swelling,inflammation, and pain. Subject was treated with the botanical blendshown in Table 3 and monitored for healing and pain levels. Thephotographs illustrate the visual symptoms at days 1 (FIG. 41 A), 6(FIG. 41B) and 9 (FIG. 41C).

Although limited to only two subjects, subjects infected withEpstein-Barr virus have also been treated. Epstein-Barr virus belongs tothe herpes virus family, but is classified as a gamma herpes virus. Itis the causative agent of infectious mononucleosis and other diseases.Subjects treated included a subject diagnosed with hairy leukoplakia.This presented as painful lesions along the tongue of this subject. Theaqueous botanical blend (no VERSABASE gel) was administered as a mouthrinse (not to be swallowed) every 4 hours. Within 4 days of treatment,lesions had healed.

Infections with alpha herpes viruses are typically associated with painand discomfort at the lesion sites. Following treatment of patients forHSV1, HSV2, and VZV, it was noted that virtually all patients expresseda rapid decrease in pain and discomfort associated with their lesions.This activity is likely associated with analgesic (anti-pain) andantipruritic (anti-itch) properties and component(s) present in the S.purpurea botanical present in the botanical extract composition. Thisproperty of S. purpurea has previously been reported in regards to otherforms of pain. The mechanism of action regarding this analgesic propertyis unknown, but adds to the therapeutic value of the botanical extractcomposition by providing pain relief along with subsequent killing ofthe viral pathogen

(HSV): Patient is 28 year old female whose chief complaint was herpessimplex I and/or II outbreak vaginally and orally. Patient history waspositive for exposure to herpes simplex I via rape. Blood work waspositive for herpes simplex I however, type II is also possible. Patienthad one vaginal sore that was positive for sharp infernal pain, dullaching, and surface tenderness and complained of “tingling, burning, anditching” of labia. Patient was given a botanical extract composition(30%) in VERSABASE gel (70%) and was instructed to apply every two hoursto infected areas after rinsing the area or douching with warm water.Patient reported initial “warm, tingling sensation” upon application oftreatment which subsided after a “few minutes.” The patient's labialsymptoms subsided within one day and non-visible by the end of the thirdday of application. Patient's vaginal pain subsided within two days andthe sore erythema decreased each day.

(VZV): Patient was a 42 year old, male. Patient presented with lesionson left side of nose and orbital region. Patient diagnosed with herpeszoster. Patient initially prescribed acyclovir. Patient also beganregime of a botanical extract composition (30%) in VERSABASE gel (70%)applied twice. Within a few hours of treatment, patient reported painwas reduced from 8/10 to 5/10. Previously patient was unable to sleep.Pain relief allowed patient to sleep. After 3 days of treatment, lesionsize had reduced and pain reduced to 3/10. After 6 days, pain reduced to1/10 and scabbing of lesions on left nostril. After nine days, lesionshave completely resolved.

Results from this work clearly demonstrate that extracts from S.purpurea can indeed inhibit the replication of various members of thepoxvirus family, including vaccinia virus, monkeypoxvirus (an emerginghuman pathogen), variola virus (the causative agent of smallpox) andlikely molluscum contagiosum. In further characterization of thebotanical extract blend, research presented herein has shown that thisextract can inhibit the replication of other, non-related viruses,including members of the herpes virus family, and the papilloma andpolyomavirus families. These pathogens are responsible for millions ofinfections every year, including cold sores, genital lesions, shingles,warts, cervical cancer, oral/throat/neck cancers, anal cancer, and maybe associated with actinic keratosis and squamous cell carcinoma. Insupport of this, experimental results demonstrate that S. purpureaextracts can be used as a therapeutic for infections caused by theseviruses as well as cervical dysplasia, oral, throat and neck cancers,squamous cell carcinoma and the pre-cancerous lesions associated withactinic keratosis.

Therapeutic Composition for Treatment of Cervical Dysplasia

A modification of this blend has been used for the treatment of cervicaldysplasia associated with HPV infection. This final blend is initiallyprepared as a aqueous (liquid) blend of the six botanical extractscontaining 83% v/v Sarracenia purpurea (125 viral inhibitory units(VTU)/ml; ranging from 60-300 VIU/ml), 7% v/v Melissa officinalis (2000VIU/ml; ranging from 1000-4000 VIU/ml), 2% v/v Lavandula officinalis(150 VIU/ml; ranging from 75-300 VIU/ml), 2% v/v Glycyrrhiza glabra (100VIU/ml; ranging from 50-500 VIU/ml), 2% v/v Eleutherococcus senticosus(500 VTU/rat, ranging from 100-2000 VIU/ml), and 4% v/v Hypericumperforatum (2000 VIU/ml; ranging from 1000-4000 VTU/ml) (FIG. 29b ).Following preparation of the aqueous (liquid) blend, this solution issubsequently combined in a 60:40 ratio (vol:wt) of aqueous blend toVERSABASE gel to formulate the final application product. Variations inthe percentages of the botanicals within this blend can be done toincrease synergistic activity and efficacy.

Methods for viral efficacy studies and botanical extract standardizationto antiviral activity

For standardization of antiviral activity associated with the botanicalextracts, the following methodology was used for HSV1:

-   -   Vero cells were grown to 90-100% confluency in Dulbecco's MEM        media containing 10% heat inactivated fetal bovine serum and        anti-fungal/anti-bacterial at 37° C. in a 5% CO₂ growth        incubator to produce cell monolayers.    -   Viral stocks were diluted to prepare aliquots containing 200        plaque forming units (pfu) in 100 μl media    -   Botanical extracts were prepared as described herein and        filtered through 0.2 μm syringe filters. Ethanol-based extracts        were vacuum evaporated for 45 minutes at room temperature.    -   Serial dilutions of botanical extracts were prepared in 2-fold        concentration steps ranging from 0.1 to 32 μl/ml, and each        botanical extract concentration was added to a viral stock        aliquot and incubated at room temperature for 20 minutes.    -   Treated viral samples were then added to the cell monolayers and        incubated with occasional rocking for 30 minutes.    -   After this infection step, fresh medium containing 0.3% human        gamma globulin and 0.9% agar was added to the cell monolayers        containing an equivalent concentration of the botanical as that        added to the viral stock aliquot tubes.    -   The infected/treated cell monolayers were incubated at 37° C. in        a 5% CO₂ growth incubator.    -   After 3 days, the agar medium was removed and the cell        monolayers stained with crystal violet to visualize plaque        formation.    -   Plaque numbers were calculated and graphed.

For other viruses, the same protocol was followed with the followingvariations: For HSV2:

For Step 8, cells were grown for 2 days, rather than 3 days. For VZV:

For Step 6, fresh medium did not contain human gamma globulin.

For Step 8, crystal violet staining was done at 9 days post infection,rather than at 3 days. For SV-40:

For Step 1, BSC-1 cells were used instead of Vero cells. Medium was MEMwith 10% heat inactivated fetal bovine serum with anti-fungal andanti-bacterial additives.

Following infection in Step 5, the plates were overlayed with mediumcontaining 0.9% agar. The plates were fed with fresh medium every 3-4days.

For Step 8, staining with crystal violet after removal of the agarmedium overlay was performed 7-10 days post infection.

For poxviruses:

For Step 6, medium did not contain human gamma globulin. S. purpurearegulation of inflammation and pro-inflammatory cytokines

In the present research, herpes virus infected subjects presentedoutbreak signs of infection including the afflicted area becomingreddened, itching and swollen. These symptoms are associated with thebody's inflammatory response towards the virus infection. Followingapplication of the botanical extract composition or Sarracenia purpureaalone, subjects typically reported a decrease in pain, redness andswelling within a few to 24 hours post application. These resultssuggest that S. purpurea components are diminishing the inflammatoryresponse to the infection. Since inflammation is associated with aninduction of pro-inflammatory cytokines, these results may suggest thatS. purpurea is capable of decreasing the expression of these keycytokines.

To confirm S. purpurea was able to regulate the expression ofpro-inflammatory cytokines, an in vitro assay was developed. Peripheralblood mononuclear cells (PBMCs) were isolated from two different healthysubjects. For immune stimulation, cells were treated with phorbol12-myristate 13-acetate (PMA) plus ionomycin (50 ng/ml and 1/ml,respectively) or lipopolysaccharide (LPS) (100 ng/ml). Treatmentsincluded the following combinations

PMA+ionomycin for 2 hours then washed

LPS for 2 hours then washed

S. purpurea extract for 4 hours followed by PMA+ionomycin for 2 hoursthen washed

S. purpurea extract for 4 hours followed by LPS for 2 hours then washed

S. purpurea extract for 4 hours then washed

Untreated

Following 6, 12 and 24 hours post treatment, total RNA was purified fromthe cells as per manufacturer's recommended protocol (Qiagen RNAisolation kit). The mRNA expression level of various cytokines wasmeasured by Real-Time PGR analysis. Real-time PGR primers and reagentswere obtained commercially and analyzed under the following PGR.conditions:

Heat to 95° C. for 10 minutes

Heat to 95° C. for 15 seconds

Heat to 40° C. for 40 seconds

Read the fluorescence level

Heat to 72° C. for 30 seconds

Repeat steps 2-5 for 44 cycles

Cytokine primers included: CCL3 (MIP-1α, Macrophage inflammatoryprotein-1α), TNF-α (Tumor necrosis factor-α), IL8 (Interleukin-8), IL5(Interleukin-5), 1L1ß (Interleukin-1 ß), IL2 (Interleukin-2), IL10(Interleukin-10), IFN-γ (interferon-γ), IL6 (Interleukin-6).

The results of these assays are shown in FIGS. 42A-42R. Cycle threshold(Ct) is the cycle number at which the fluorescence for the reactioncrosses the threshold value. In this figure, the ΔCt value representsthe change in the Ct value of the experimental group minus the untreatedgroup. As shown, PMA+ionomycin and LPS treatment induced the expressionof various cytokines as represented by a decrease in the ΔCt value. Thiscan easily be seen for CCL3 (FIGS. 42A and 42B (subject 1 and subject 2,respectively)), TNF-α (FIGS. 42C and 42D (subject 1 and subject 2,respectively)), IL5 (FIGS. 42K and 42L (subject 1 and subject 2,respectively)), IL-1B (for LPS) (FIGS. 42E and 42F (subject 1 andsubject 2, respectively)), IL2. (for PMA+ionomycin) (FIGS. 42G and 42H(subject 1 and subject 2, respectively)), IFN-γ (FIGS. 42O and 42P(subject 1 and subject 2, respectively)), and IL6 (FIGS. 42M and 42N(subject 1 and subject 2, respectively)). Cells which were pre-treatedwith S. purpurea followed by PMA+ionomycin or LPS, typically showed adecrease in the expression level of these cytokines. This can beobserved especially for CCL3, IL5 (for PMA-ionomycin), IL-1 ß, and IL6(FIGS. 42Q and 42R (subject 1 and subject 2, respectively)). Inaddition, treatment of cells with S. purpurea extract alone led to adecrease in the endogenous level of expression of various cytokines.This can be observed especially for CCL3, TNF-α, IL8 (FIGS. 42I and 42J(subject 1 and subject 2, respectively)), IL-1 ß, ILK) (FIGS. 42M and42N (subject 1 and subject 2, respectively)), and IL6.

For the data illustrated in FIGS. 43A-44I, specific time points weregraphed showing the fold-change of expression of the cytokine mRNAsrelative to untreated cells. As shown in FIGS. 43A-43L S. purpureatreatment led to a block in cytokine induction by PMA+ionomycin for CCL3(18 HPT, FIG. 43A), TNF-α (12 HPT, FIG. 43B), IL1 ß (12 HPT, FIG. 43C),IL2 (6HPT, FIG. 43D), IL5 (16 HPT, FIG. 43F), IL10 (6HPT, FIG. 43G),IFN-γ (12 HPT, FIG. 43H) and IL6 (12 HPT, FIG. 43I). Notably, no changewas observed for IL8 (12 HPT, FIG. 43E). Similar results were observedfor the results of the experiment illustrated in FIGS. 44A-44I where S.purpurea treatment led to a block in cytokine induction by LPS for CCL3(18 HPT, FIG. 44A), TNF-α (12 HPT, FIG. 44B), IL-1 β (12 HPT, FIG. 44C),IL2 (6HPT, FIG. 44D), ILK), (6HPT, FIG. 44G) IFN-γ (12 HPT, FIG. 44H)and IL6 (12 HPT, FIG. 44I). Notably, only minor changes were observedfor IL8 (12 HPT, FIG. 44E) and IL5 (16 HPT, FIG. 44F).

Together, these in vitro results support results observed in humansubjects treated with a botanical extract composition in accordance withvarious embodiments, or S. purpurea extract alone, where a reduction ininflammation was observed. These results support that S. purpurea canreduced the expression of various cytokines involved in apro-inflammatory response.

Comparison of Ethanol and Glycerin Based Extraction Solvents

FIGS. 45A-45F illustrate the results of comparison of the activity ofthe six Species II extracts for ethanol versus glycerin based extractionmethods. As shown, for five of the six Species II botanical extractsdescribed, extractions can be performed either using ethanol-basedsolutions or glycerin-based solutions, with the extracts showing similarantiviral activity. Ethanol-based extractions were performed withethanol percentages as described in the Extract Preparation section.Glycerin extraction was done using distilled water/glycerol (25%/75%).As shown, anti-viral activity associated with S. purpurea (FIG. 45A), M.officinalis (FIG. 45E), G. glabra (FIG. 45B), E. senticosus (FIG. 45F)and H. perforatum (FIG. 45C) was similar whether extracted using ethanolor glycerin based extraction solutions. Notably, extraction of L.officinalis (FIG. 45D) required extraction using a glycerin basedextraction solution. Extraction of this botanical with an ethanol basedsolution did not produce any detectable antiviral activity. Theseresults suggest that the active constituents) present in Lavandulaofficinalis requires the presence of glycerin to either release theactive molecules from the plant material or to maintain the activeconstituent(s) in a stable, active form.

Procedure for Extraction of Sarracenia Species Plant Material

Finely ground dried plant material was mixed with extraction solution ata ratio of 1:15 kg plant material:liter extraction solution. Extractionsolution was 5:4:1 190 proof ethanol:distilled water:glycerin. Themixture was incubated at room temperature in an amber glass container,with agitation for 1-2 minutes every 6 hours to resuspend solidmaterial. After a 7 day extraction period, the extraction mixture wasclarified by centrifugation, and the solid cake discarded. The clarifiedextraction was then filtered through a 0.2 μm filter into a sterilecontainer.

Procedure for Extraction of Melissa officinalis Species Plant Material

Finely ground dried plant material was mixed with extraction solution ata ratio of 1:8 kg plant material: liter extraction solution. Extractionsolution was 3:1 glycerin:distilled water. The mixture was incubated atroom temperature in an amber glass container, with agitation for 1-2minutes every 6 hours to resuspend solid material. After a 7 dayextraction period, the extraction mixture was clarified bycentrifugation, and the solid cake discarded. The clarified extractionwas then filtered through a 0.2 μm filter into a sterile container.

Procedure for Extraction of Lavandula officinalis Species Plant Material

Finely ground dried plant material was mixed with extraction solution ata ratio of 1:8 kg plant material: liter extraction solution. Extractionsolution was 3:1 glycerin:distilled water. The mixture was incubated atroom temperature in an amber glass container, with agitation for 1-2minutes every 6 hours to resuspend solid material. After a 7 dayextraction period, the extraction mixture was clarified bycentrifugation, and the solid cake discarded. The clarified extractionwas then filtered through a 0.2 μm filter into a sterile container.

Procedure for Extraction of Hypericum perforatum Species Plant Material

Finely ground dried plant material was mixed with extraction solution ata ratio of 1:4 kg plant material: liter extraction solution. Extractionsolution was 58:32:10 ethanol:distilled water:glycerin. The mixture wasincubated at room temperature in an amber glass container, withagitation for 1-2 minutes every 6 hours to resuspend solid material.After a 7 day extraction period, the extraction mixture was clarified bycentrifugation, and the solid cake discarded. The clarified extractionwas then filtered through a 0.2 μm filter into a sterile container.

Procedure for Extraction of Eleutherococcus senticosus Species PlantMaterial

Finely ground dried plant material was mixed with extraction solution ata ratio of 1:6 kg plant material: liter extraction solution. Extractionsolution was 42:53:5 ethanol:distilled water:glycerin. The mixture wasincubated at room temperature in an amber glass container, withagitation for 1-2 minutes every 6 hours to resuspend solid materialAfter a 7 day extraction period, the extraction mixture was clarified bycentrifugation, and the solid cake discarded. The clarified extractionwas then filtered through a 0.2 μm filter into a sterile container.

Procedure for Extraction of Glycyrrhiza glabra Species Plant Material

Finely ground dried plant material was mixed with extraction solution ata ratio of 1:6 kg plant material: liter extraction solution. Extractionsolution was 3:1 glycerin:distilled water. The mixture was incubated atroom temperature in an amber glass container, with agitation for 1-2minutes every 6 hours to resuspend solid material. After a 7 dayextraction period, the extraction mixture was clarified bycentrifugation, and the solid cake discarded. The clarified extractionwas then filtered through a 0.2 μm filter into a sterile container.

Compositions

TABLE 5 Component concentrations of manufactured 100 ml botanicalextract composition. VIU/ml VIU/100 ml Percent botanical BatchIngredient (Vol/Vol) extract LIQUID blend Sarracenia purpurea 50 1253250 Melissa officinalis 12 2000 24000 Lavendula officinalis 20 150 3000Glycyrrhiza glabra 5 100 500 Hypericum performatum 5 2000 10000Eleutherococcus senticosus 8 500 4000 Total 100 N/a 47750

TABLE 6 Therapeutic composition formulation. VIU/100 ml Batcj IngredientPercent GEL blend Botanical extract composition 50 (vol.) 47750 (fromTable 5) Versa Base gel 50 (wt.) None* Total 100 23875

All publications, patents and patent applications are incorporatedherein by reference. While in the foregoing specification this inventionhas been described in relation to various embodiments thereof, and manydetails have been set forth for purposes of illustration, it will beapparent to those skilled in the art that the invention is susceptibleto additional embodiments and that certain of the details describedherein may be varied considerably without departing from the basicprinciples of the present disclosure.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention are to be construed to cover boththe singular and the plural, unless otherwise indicated herein orclearly contradicted by context. The terms “comprising,” “having,”“including,” and “containing” are to be construed as open-ended terms(i.e., meaning “including, but not limited to”) unless otherwise noted.Recitation of ranges of values herein are merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range, unless otherwise indicated herein, and eachseparate value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g., “such as”) provided herein, isintended merely to better illuminate the invention and does not pose alimitation on the scope of the invention unless otherwise claimed. Nolanguage in the specification should be construed as indicating anynon-claimed element as essential to the practice of the invention.

Embodiments of this invention are described herein, including the bestmode known to the inventors for carrying out the invention. Variationsof those embodiments may become apparent to those of ordinary skill inthe art upon reading the foregoing description. The inventors expectskilled artisans to employ such variations as appropriate, and theinventors intend for the invention to be practiced otherwise than asspecifically described herein. Accordingly, this invention includes allmodifications and equivalents of the subject matter recited in theclaims appended hereto as permitted by applicable law. Moreover, anycombination of the above-described elements in all possible variationsthereof is encompassed by the invention unless otherwise indicatedherein or otherwise clearly contradicted by context.

What is claimed is:
 1. A method for inhibiting a replication of apoxvirus by exposing the poxvirus to a liquid extract from a plantmaterial selected from Sarracenia, Nepenthes, Melissa, Lavandula,Glycyrrhiza, Eleutherococcus, Hypericum, Darlingtonia, Heliamphora,Roridula, Drosera, Dionaea, Aldrovanda, Drosophyllum, Triphyophyllum,Catopsis, Brocchinia, Paepalanthus, Uricularia, Genlisea, Pinguicula,Ibicella, Byblis, Philcoxia, Stylidium and Cephalotus wherein the plantmaterial is prepared by steps comprising: obtaining fresh plant materialless than about ten (10) days following harvest of the plant material;washing and air drying the plant material; combining the plant materialwith a liquid comprising ethanol and glycerol at a ratio of about one(1) gram of the plant material to about fifteen (15) milliliters of theliquid; allowing the liquid to extract the plant material at roomtemperature in an amber glass jar for about two (2) to about sixty (60)days or by boiling for between about thirty (30) minutes to about four(4) hours to form the liquid extract; and separating the liquid extractfrom the plant material.
 2. The method of claim 1 wherein the poxvirusis selected from the group consisting of orthopoxvirus, parpoxvirus,avipoxvirus, capripoxvirus, leporipoxvirus, suipoxvirus,molluscipoxvinis molluscipox virus and yatapox virus.
 3. The method ofclaim 1, wherein the pox virus is selected from the group consisting ofvaccinia virus, monkeypox virus, variola virus, and molluscumcontagiosum virus.
 4. The method of claim 1, further comprising stepsstandardizing a biological activity of the liquid extract comprising:growing a cell culture to a target cell density for a viral infection;preparing a dilution of a viral stock to a known plaque forming unit(pfu) concentration; preparing a serial dilution of the liquid extractto produce a plurality of liquid extract treatment concentrations;combining the dilution of the viral stock with each of the plurality ofliquid extract treatment concentrations; adding each of the plurality ofliquid extract treatment concentration/viral stock combinations to analiquot of the cell culture to produce a plurality of infected cellcultures; adding a fresh equivalent liquid extract treatmentconcentration to each of the plurality of infected cell cultures;incubating each of the plurality of the infected cell culture/liquidextract combinations; measuring a viral plaque formation by counting thenumber of viral plaques and reporting the viral plaque formation as apercentage of plaque formation, wherein the percentage of plaqueformation is the observed number of plaques divided by an expectednumber of plaques based on the viral concentration of the viral stockaliquot; and determining the biological activity of the liquid extractfrom the percentage of viral plaque formation.
 5. The method of claim 4,wherein the liquid extract comprises about 50% of an extract ofSarracenia purpurea having biological activity of about fifty (50) viralinhibitory units per milliliter and about 12% of an extract of Melissaofficinalis having a biological activity of about eighty (80) viralinhibitory units per milliliter.